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.).

실험 1. 계획 <ol><li> 합성에 필요한 구리 나노 복합 재료의 양을 결정합니다. 그 기준으로, 작은 볼륨 플라스크 (25cm 2), 또는 자료의 준비에 아래에 표시된 바와 같이 더 큰 플라스크의 번호를 선택합니다. </li><li> 이 합성에 대해, 5 % CO 2와 적어도 40 %의 습도와 37 ° C의 배양기를 사용한다. 이러한 보육이 가능하며 반복적으로 합성의 시간 (약 24 시간) 이상 방해되지 않습니다 있는지 확인하십시오. 주의 : 반복 열기 및 인큐베이터의 폐쇄 확실히 나노 복합 구조의 변형 합성 될 수 있습니다 온도 변화의 원인이됩니다. </li></ol> 재료 2. 준비 <ol><li> 합성을 시작하는 것입니다 직전 용매에 고체 물질을 추가하여, 실험 시작하기 전에 신선한 모든 재료를 준비합니다. 긴 시간 B에 대한 액체에 시스틴과 구리 출발 물질의 재고 솔루션을 유지는 efore 실험은 권장되지 않으며 변수의 결과가 발생할 수 있습니다. 일단 파라 필름과 용기의 상단을 포장하여 건조 원료 공급 업체에서 계속 열었다. 주 : 다음 프로토콜 시스틴 7 μL, 멸균 6643 μL 및 CNPS 350 μL를 사용하여 25cm 2 세포 배양 플라스크 내의 반응 예로서 사용된다. </li><li> CNPS의 최소 2 mg의를 무게에 의해 구리 나노 입자의 2 ㎎ / ㎖ 솔루션을 준비합니다. 피부에 CNPS 가능한 접촉을 방지하기 위해이 단계에서 일회용 장갑을 착용 할 것. 빈 멸균 16 mL 유리 병에 나노 입자를 놓습니다. <ol><li> CNPS이 들어있는 유리 병에, 2 ㎎ / ㎖ 용액을 합성 시작하기 전에 나노 입자의 분산을 제공하기 위해 20 초 동안 솔루션을 와동 적절한 볼륨에서 멸균 탈 이온수를 추가 (최소 1 ml의 총 부피 권장). 이 소용돌이로 교반하여 혼합 억제하므로 물을 유리 병의 절반 이상이 방법을 기입하지 마십시오. CNPS 줘야난 빠르게 유리 병의 바닥에 정착 (검정 회색) 색상 어두운 나타납니다. </li><li> 합성의 시작하기 전에 CNPS의 최대 분산을 제공하기 위해 실온에서 17 분 동안 CNP 솔루션을 sonicate하세요. 주기적으로 CNPS은 초음파로 인해 혼합되어 있는지 확인하십시오. 성공적인 초음파 처리 후, CNPS 적어도 30 분 동안 용액에 현탁 상태로 유지하고, 용액의 색은 어두운 것이다. </li></ol></li><li> 합성 72.9 ㎎ / ㎖ 용액을 만들기 위해 충분한 시스틴의 질량을 단다. 시스틴 물에 직접 용해하지 않기 때문에, 대전 계량 용기에 칭량 시스틴 놓는다. <ol><li> 시스틴이 완전히 용해되도록 계량 용기 포함 시스틴에, 멸균, 1 M NaOH를 충분한 볼륨을 추가합니다. 예를 들어, 72.9 ㎎ / ㎖ 용액을 만들기 위해, 1 M NaOH를 100 μL 완전히 시스틴 7.29 mg을 용해. </li><li> 멸균 흐름 조직 문화 후드에서이 단계를 수행, 무균 상태를 유지합니다. 주의 : 수산화 나트륨1 M 농도가 가성이다에서, 그래서 피부에 농축 된 수산화 나트륨의 접촉을 방지하기 위해이 단계에서 일회용 장갑을 착용 </li></ol></li><li> 무균 조직 배양 후드에서 작업, 먼저 멸균 합성 플라스크에 멸균의 6643 μL과 시스틴의 7 μl를 추가하고, 플라스크 캡으로 37 ° C에서 인큐베이터에서 30 분 동안 품어 보자 효과 제공 (느슨한) 배출 혼합. CNPS는 초음파 단계 후에 정착하기 때문에, 30 초 동안 볼 텍싱하여 2 ㎎ / ㎖ 용액을 CNP 재현 탁. <ol><li> 합성을 시작하도록 25cm 2 세포 배양 플라스크에 멸균 수, 50 부 CNPS 및 949 부를 1 부품 시스틴 결합 : 다음의 성분 비율을 유지하기 위해 (무균 기술을 사용하여) 합성 플라스크에 충분한 CNP 용액을 추가한다. 예를 들어, 7 mL의 합성 볼륨, 시스틴 원액 7 μL, CNPS 350 μL, 멸균 물 6643 μL를 결합한다. 플라스크에 뚜껑을 교체하고 그래서 SECUR가 있음을 조전자. </li><li> 합성을위한 모든 구성 요소를 조합 한 후, 가볍게 4-5 회 소용돌이에 의해 플라스크에 혼합한다. CO 2 배양기에서 플라스크를 놓고와 합성 동안 플라스크 중 가스 교환이 될 수 있도록 뚜껑을 풀어 플라스크를 배출. </li></ol></li><li> 합성이 약 24 시간 동안 인큐베이터에서 실행할 수 있습니다. 합성 중 하나는, 현미경과 눈으로, 높은 선형 복합의 형성을 관찰 할 수있다. 주 : 구조체의 형성 과정은 구조가 초기에 감지하기 어려운 것을 의미에서 갑자기 발생할 수 후 외관 밀도 증가에 신속하게 진행한다. 형성은 따라서 24 시간 전에 발생할 수 있습니다. 큰 구조 및 그 밀도가 증가하게되면 프로세스는 육안으로 관찰 될 수있다. 구조물의 생성 이후 시점에서 현미경으로 눈에 의해 시간이 지남에 따라 관찰 될 수 있지만, 연속 합성 조건을 중단하고 온도 불량으로 이어질합성 결과. </li><li> 단단히 합성 플라스크 상한 및 냉장고 (4 ℃)에서 선박을 저장하여 biocomposites의 합성을 종료합니다. 한번 생성 된 구조는 1 년 이상이 형태로 안정적으로 유지. 사용되는 구성 요소, 합성의 날짜 및 종료 전에 합성의 배양 시간을 포함하여 합성 조건과 플라스크 레이블. </li></ol> 구리 황산을 사용하여 3. 합성 <ol><li> 황산구리 염 CNPS 대체하여 자기 조립 합성을 수행. 멸균 기법을 사용하여, 2 ㎎ / ㎖ 용액을 멸균 증류수 충분한 부피 황산구리 적어도 2 mg을 용해. 황산동 결정을 쉽게 농도로 용액에 들어가 있지만, 필요한 경우 바이알 와동 모든 결정체가 용해되어 육안으로 확인하기 위해 검사한다. </li><li> 전술하지만 황산구리 CNPS 대체로서 황산동의 제조 후, 합성을 수행한다. 주 : 출발 물질로서 황산 구리를 사용하여 자기 조립 된 나노 복합 재료가 CNPS 합성 구조물의 최종 형상보다 훨씬 더 일관되게 나타났다. </li><li> CNP의 복합 재료로 구리 황산 biocomposites의 합성을 종료 (2.6 단계), 4 ℃에서 그들에게 장기를 저장합니다. </li></ol> 4. 특성화 및 Biocomposites 후 합성 처리 <ol><li> CNPS에서 흰색 빛 현미경 (9)에 의해 구리 황산에서와 전자 현미경 (9)에 의해 유도 된 biocomposites을 특성화. <ol><li> 합성 플라스크 평 누워의 몇 분 이내에 플라스크의 바닥 표면에 정착하는 바와 같이 특성화 및 biocomposites 백색광 현미경으로 합성 후 검사를 들어, 거꾸로 현미경을 사용하여, 다음 포커스하게 할 수있다. biocomposites 및 액체 매질 사이의 대비를 극대화하기 위해 현미경의 시야 설정을 사용합니다. CNPS과 경찰에서 파생 된 복합황산 당 모두 색상의 불투명 분명 나타날 것입니다 만, 미 반응 CNP 집계 색상에 매우 어두운 나타납니다. <ol><li> 복합 이미지를 캡처하기 위해 현미경에 연결된 디지털 카메라를 사용한다. 개별 구조물의 길이의 범위가 관찰됩니다. </li></ol></li><li> 특성화 및 biocomposites 합성 후 검사의 경우 4 ℃에서 저장 한 후, 플라스크 현미경 이미징을 수행하는 동안 집중 효과가 모호합니다 냉장고에서 제거에 처음 응축을 형성하므로 플라스크 적어도 15 분 동안 실온으로 올 수 있습니다. RT로 평형을 허용 한 후, 현미경 이미징 품질을 최대화하기 위해 깨끗한 종이 타월로 플라스크의 상부 및 하부 표면을 닦아. </li><li> 냉장고에있는 동안 형성 복합 재료의 덩어리를 떼어 놓다 30 초 동안 장기 저장되어있는 또는 영상 복합, 소용돌이 플라스크를 작업 할 때. 텍싱 후, 반전 마이크와 구조를 검사roscope는 집계가 해리했는지 확인하고 필요 텍싱 반복합니다. </li><li> CNPS를 사용하여 주어진 실험 합성의 효능을 평가하기 위해 반전 된 백색광 현미경을 사용한다. 예를 들어, 합성시와 같은 다른 파라미터를 가지는 플라스크에서 CNP 유래 biocomposites 사용 합성 플라스크 CNPS 미 반응의 유무를 기록. 개별 CNPS는 광학 현미경으로 관찰하기에 너무 작지만 미 CNP은 높은 종횡비, 선형 형태를 가질 것이다 성공적으로 합성 CNP 합성물 달리, 둥근 모양과 어두운 객체로 나타난다 집계 한 참고 서로 다른 길이의 범위를해야합니다. 이것이 일단 형성되면 개별 구조로 분산시키는 것이 곤란하다 고도로 분지 &quot;게&quot;형 구조물을 초래할 것 같은 종료 전에 일정 기간 너무 오랫동안 합성을 수행 피한다. </li><li> 효능을 평가하기 위해 반전 하얀 빛 현미경을 사용하여황산구리를 사용하여 주어진 실험 합성. 황산동이 프로토콜을 이용하여 용액에 완전히 나가기 때문에, 용액을 사용 CNPS 합성 용액으로부터보다 어둡게 나타날 것이다. 이러한 종료​​ 전에 합성 시간으로서 다른 합성 조건에 플라스크를 비교하여 황산구리 복합체의 크기와 정도를 문서화. 참고 : 성공적으로 합성 된 복합 재료가 서로 다른 길이의 범위를 표시합니다. 이 &quot;성게 같은&quot;구조 될 것입니다 일부 복합의 높은 분기 집계 결과 바와 같이, 종료 전에 시간의 기간이 너무 오래 합성을 수행하지 않도록하고있는 일단 형성되면 개별 구조로 분산하기가 어렵습니다 . </li></ol></li><li> biocomposites를 합성 후, 원심 분리 튜브에서 원심 복합 솔루션을 집중. 15 ㎖의 원심 분리 관에 CNP 유래 구조 또는 구리 황산 파생 구조 중 6 ML을 추가합니다. 10 분 동안 원심 분리기RT 500 XG에 펠릿을 형성 하였다. 작은 볼륨의 경우, 0.6 ml의 크기의 튜브에 솔루션 구조의 500 μl를 추가합니다. 적어도 10 분 동안 실온에서 2000 XG에 원심 펠렛을 형성 하였다. <ol><li> 충분한 시간 동안 (microfuges 적어도 10 분)을 원심 분리 한 후, 구조가 심하게 펠릿 상기 상등액을 제거하여 농축 튜브의 하단에 관찰 된 펠릿을 저장한다. 구리 황산에서 파생 Biocomposite 구조는 색상과 CNPS에서 파생 된 구조의 청색 어두운 (검정 회색)되어 나타납니다. </li><li> 이 튜브에 더 많은 복합 재료를 추가하고 필요한 경우 구조를 집중하기 위해 동일한 튜브의 과정을 반복한다. 농축 된 알약을 분산하기 위해 10 ~ 30 초 동안 원하는 튜브 용액의 부피, 소용돌이를 추가합니다. </li></ol></li><li> 초음파 처리 구조는 한번 값을 낮추는 구조의 인구 평균 크기 (길이)를 이동하도록 형성되어있다. 르 멸균 탈 이온수 및 초음파 처리에 넣어 구조AST 10 분. 시간이 지남에 따라이 공정을 이용하여, 구조가 단편화 및 평균 길이 (텍스트도 6 참조)에서 작아진다. 역 하얀 빛 현미경과 디지털 카메라를 사용하여 서로 다른 초음파 시간과 복합 크기의 문서 변경. </li></ol>

<ol>
	<li>Klinman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+copper-enzyme+family+of+dopamine+beta-monooxygenase+and+peptidylglycine+alpha-hydroxylating+monooxygenase:+resolving+the+chemical+pathway+for+substrate+hydroxylation.">The copper-enzyme family of dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation.</a> The Journal of biological chemistry. 281, 3013-3016 (2006).</li><li>Uauy, R., Olivares, M., Gonzalez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentiality+of+copper+in+humans.">Essentiality of copper in humans.</a> The American journal of clinical nutrition. 67, 952S-959S (1998).</li><li>Karlsson, H. L., Cronholm, P., Gustafsson, J., Copper Moller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=oxide+nanoparticles+are+highly+toxic:+a+comparison+between+metal+oxide+nanoparticles+and+carbon+nanotubes.">oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes.</a> Chemical research in toxicology. 21, 1726-1732 (2008).</li><li>Parekh, G., 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=Layer-by-layer+nanoencapsulation+of+camptothecin+with+improved+activity.">Layer-by-layer nanoencapsulation of camptothecin with improved activity.</a> International journal of pharmaceutics. 465, 218-227 (2014).</li><li>Harrington, M. J., Masic, A., Holten-Andersen, N., Waite, J. H., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-clad+fibers:+a+metal-based+biological+strategy+for+hard+flexible+coatings.">Iron-clad fibers: a metal-based biological strategy for hard flexible coatings.</a> Science. 328, 216-220 (2010).</li><li>Keyson, D., 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=CuO+urchin-nanostructures+synthesized+from+a+domestic+hydrothermal+microwave+method.">CuO urchin-nanostructures synthesized from a domestic hydrothermal microwave method.</a> Materials Research Bulletin. 43, 771-775 (2008).</li><li>Liu, B., Zeng, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+organization+of+CuO+nanoribbons:+formation+of+'dandelions'.">Mesoscale organization of CuO nanoribbons: formation of 'dandelions'.</a> J Am Chem Soc. 126, 8124-8125 (2004).</li><li>Peng, 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=Controllable+synthesis+of+self-assembled+Cu2S+nanostructures+through+a+template-free+polyol+process+for+the+degradation+of+organic+pollutant+under+visible+light.">Controllable synthesis of self-assembled Cu2S nanostructures through a template-free polyol process for the degradation of organic pollutant under visible light.</a> Materials Research Bulletin. 44, 1834-1841 (2009).</li><li>Deodhar, S., Huckaby, J., Delahoussaye, M., DeCoster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Aspect+Ratio+Bio-Metallic+Nanocomposites+for+Cellular+Interactions.">High-Aspect Ratio Bio-Metallic Nanocomposites for Cellular Interactions.</a> IOP Conference Series: Materials Science and Engineering. 64, 012014 (2014).</li><li>Montes-Burgos, I., 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=Characterisation+of+nanoparticle+size+and+state+prior+to+nanotoxicological+studies.">Characterisation of nanoparticle size and state prior to nanotoxicological studies.</a> Journal of Nanoparticle Research. 12, 47-53 (2010).</li><li>Wiogo, H. T., Lim, M., Bulmus, V., Yun, J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+magnetic+iron+oxide+nanoparticles+in+biological+media+by+fetal+bovine+serum+(FBS).">Stabilization of magnetic iron oxide nanoparticles in biological media by fetal bovine serum (FBS).</a> Langmuir. 27, 843-850 (2011).</li><li>Yunker, P. J., Still, T., Lohr, M. A., Yodh, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+the+coffee-ring+effect+by+shape-dependent+capillary+interactions.">Suppression of the coffee-ring effect by shape-dependent capillary interactions.</a> Nature. 476, 308-311 (2011).</li><li>Kahler, H., Lloyd Jr, B., Eden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Microscopic+and+Other+Studies+on+a+Copper&#8211;Cystine+Complex.">Electron Microscopic and Other Studies on a Copper&#8211;Cystine Complex.</a> The Journal of Physical Chemistry. 56, 768-770 (1952).</li><li>Furia, E., Sindona, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+of+L-cystine+with+metal+cations.">Complexation of L-cystine with metal cations.</a> Journal of Chemical &amp; Engineering Data. 55, 2985-2989 (2010).</li><li>Hawkins, C., Perrin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polynuclear+Complex+Formation.+II.+Copper+(II)+with+Cystine+and+Related+Ligands.">Polynuclear Complex Formation. II. Copper (II) with Cystine and Related Ligands.</a> Inorganic Chemistry. 2, 843-849 (1963).</li><li>Hallman, P., Perrin, D., Watt, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+computed+distribution+of+copper+(II)+and+zinc+(II)+ions+among+seventeen+amino+acids+present+in+human+blood+plasma.">The computed distribution of copper (II) and zinc (II) ions among seventeen amino acids present in human blood plasma.</a> Biochem. J. 121, 549-555 (1971).</li><li>Jensen, L. S., Maurice, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sulfur+amino+acids+on+copper+toxicity+in+chicks.">Influence of sulfur amino acids on copper toxicity in chicks.</a> The Journal of nutrition. 109, 91-97 (1979).</li><li>Lee, Y., Choi, J. R., Lee, K. J., Stott, N. E., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+synthesis+of+copper+nanoparticles+by+chemically+controlled+reduction+for+applications+of+inkjet-printed+electronics.">Large-scale synthesis of copper nanoparticles by chemically controlled reduction for applications of inkjet-printed electronics.</a> Nanotechnology. 19, 415604 (2008).</li><li>Hume, 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=Engineered+coiled-coil+protein+microfibers.">Engineered coiled-coil protein microfibers.</a> Biomacromolecules. 15, 3503-3510 (2014).</li></ol>

Authors have nothing to disclose.

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 &quot;게&quot;를 포함하는 전술 구리 연상시키는 중앙 집합체에서 높은 선형 돌기를 형성 6. 그것은 여기에 ...

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 &#176;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 &#8220;urchin&#8221; and &#8220;nanoflower&#8221; 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 &#176;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 &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table><tbody><tr><td>Mini Vortexer</td><td>VWR (https://us.vwr.com)</td><td>58816-121</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator Model # 2425-2</td><td>VWR (https://us.vwr.com)</td><td>Contact vendor</td><td>Current model calalog # 98000-360</td></tr><tr><td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td><td>Labnet (http://labnetinternational.com/)</td><td>C2500-R</td><td>Model Prism R</td></tr><tr><td>Cell Culture Centrifuge Model Z323K</td><td>Labnet (http://labnetinternational.com/)</td><td>Contact vendor</td><td>Current model Z206A catalog # C0206-A</td></tr><tr><td>Sonicator (Ultrasonic Cleaner)</td><td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td><td>1510R-MTH</td><td></td></tr><tr><td>Balance</td><td>Sartorius (http://dataweigh.com)</td><td></td><td>Model CP225D similar model CPA225D</td></tr><tr><td>Olympus IX51 Inverted Light Microscope</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>Olympus DP71 microscope digital camera</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>external power supply unit - white light for Olympus microscope</td><td>Olympus (http://olympusamerica.com</td><td>TH4-100</td><td></td></tr><tr><td>10X, 20X, and 40X microscope objectives</td><td>Olympus (http://olympusamerica.com</td><td>Contact vendor</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td><td>model S-4800</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td><td>model Libra 120</td><td></td></tr><tr><td>Table Top Work Station Unidirectional Flow Clean Bench</td><td>Envirco (http://envirco-hvac.com)</td><td>model PNG62675</td><td>Used for sterile cell culture technique</td></tr></tbody></table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

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 제어하는​​데 도움이 입증되었다. 형성된 구조는 고도로 응집하지 않는 안정한 액상이 형성되어 있기 때문에, 원심 분리는 또한 집중 형성된 복합체 편석을 돕기 위해 사용될 수있다.

도 1은 본 연구에서 설명한 선형 biocomposites을 형성하는 합성 단계의 흐름도의 개략을 나타낸다. 출발 원료로 CNPS 또는 황산구리는 2 ㎎ / ㎖ 용액을 형성하기 위해 멸균 수와 함께,이 용액을 혼합하고 균일 혼합을 제공하는 초음파 처리하고,이 구리 용액을 합성에 대해 다음과 같은 비율로 혼합되어있다 : 949 부 멸균 물 : 50 부 구리 혼합물 : 1 부 시스틴 원액. 실제 볼륨은 확장 또는 최?...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

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.

생물 액체 배지에서 확장 성, 금속 높은 가로 세로 비율 나노 복합 재료의 생성

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.0K 조회수.  Centenary College of Louisiana.  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. 

비디오: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

액체 셀 전송 전자 현미경을 사용하여 액체의 재료의 동적 프로세스를 공개

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

연구

공학

Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding combined with layer transfer enables the replication of arbitrary surface patterns from a master structure onto the functional material. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes. In particular we demonstrate the fabrication of patterned transparent zinc oxide electrodes for light trapping applications in solar cells.

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.

기능성 재료 Nanomoulding, Nanoimprinting에 다목적 보완 패턴 복제 방법

We describe a nanomoulding technique which  allows low-cost nanoscale patterning of functional materials, materials stacks  and full devices. Nanomoulding ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

미세 유체 침투를 통해 세 가지 차원 미세 구조 나노 복합 재료의 제조

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

화학

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

영가 코어 금속 나노 입자의 생성은 사용 N- (2- 아미노 에틸) -3- aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Indirect Fabrication of Lattice Metals with Thin Sections Using Centrifugal Casting

One of the typical methods to manufacture 3D lattice metals is the direct-metal additive manufacturing (AM) process such as Selective Laser Melting (SLM) and Electron Beam Melting (EBM). In spite of its potential processing capability, the direct AM method has several disadvantages such as high cost, poor surface finish of final products, limitation in material selection, high thermal stress, and anisotropic properties of parts. We propose a cost-effective method to manufacture 3D lattice metals. The objective of this study is to provide a detailed protocol on fabrication of 3D lattice metals having a complex shape and a thin wall thickness; e.g., octet truss made of Al and Cu alloys having a unit cell length of 5 mm and a cell wall thickness of 0.5 mm. An overall experimental procedure is divided into eight sections: (a) 3D printing of sacrificial patterns (b) melt-out of support materials (c) removal of residue of support materials (d) pattern assembly (e) investment (f) burn-out of sacrificial patterns (g) centrifugal casting (h) post-processing for final products. The suggested indirect AM technique provides the potential to manufacture ultra-lightweight lattice metals; e.g., lattice structures with Al alloys. It appears that the process parameters should be properly controlled depending on materials and lattice geometry, observing the final products of octet truss metals by the indirect AM technique.

An indirect additive manufacturing method combining a 3D printing of polymers with a centrifugal casting is outlined for manufacturing 3D octet truss metals (Al and Cu alloys) having a unit cell length of 5 mm with a wall thickness of 0.5 mm.

One of the typical methods to manufacture 3D lattice metals is the direct-metal additive manufacturing (AM) process such as Selective Laser Melting (SLM) ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

모공 그라디언트, 고분자 전해질 및 그들의 간접적 인 모니터링과 금속 임플란트의 멀티 스케일 변경<em&gt; 생체 내</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

생체공학

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

화학 합성 및 시간 - 해결 기계론의 연구 Millifluidics

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Preparation of Liquid-exfoliated Transition Metal Dichalcogenide Nanosheets with Controlled Size and Thickness: A State of the Art Protocol

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. Layered crystals of molybdenum disulphide (MoS2) and tungsten disulphide (WS2) are exfoliated in aqueous surfactant solution by sonication. This yields highly polydisperse mixtures containing nanosheets with broad size and thickness distributions. However, for most purposes, specific sizes (in terms of both lateral dimension and thickness) are required. For example, large and thin nanosheets are desired for (opto) electronic applications, while laterally small nanosheets are interesting for catalytic applications. Therefore, post-exfoliation size selection is an important step that we address here. We provide a detailed protocol on the efficient size selection in large quantities by liquid cascade centrifugation and the size and thickness quantification by statistical microscopic analysis (atomic force microscopy and transmission electron microscopy). The comparison of MoS2 and WS2 shows that both materials are size-selected in a similar way by the same procedure. Importantly, the dispersions of size-selected nanosheets show systematic changes in their optical extinction spectra with size due to edge and confinement effects. We show how these optical changes are related quantitatively to the nanosheets dimensions and describe how mean nanosheets length and layer number can be extracted reliably from the extinction spectra. The exfoliation and size selection protocol can be applied to a broad range of layered crystals as we have previously demonstrated for graphene, gallium sulphide (GaS) and black phosphorus.

A protocol for the liquid exfoliation of layered materials to nanosheets, their size selection and size measurement by microscopic and spectroscopic techniques is presented.

액체 박리 전이 금속 Dichalcogenide 제어 된 크기와 나노 시트와 두께의 준비 : 예술 프로토콜의 주

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. ...

Processing of Bulk Nanocrystalline Metals at the US Army Research Laboratory

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued development of nanocrystalline metals. Despite these efforts, the transition of these materials from the lab bench to actual applications has been blocked by the inability to produce large scale parts that retain the desired nanocrystalline microstructures. Following the development of a method proven to stabilize the nanosized grain structure to temperatures approaching that of the melting point for the given metal, the US Army Research Laboratory (ARL) has progressed to the next stage in the development of these materials - namely the production of large scale parts suitable for testing and evaluation in a range of relevant test environments. This report provides a broad overview of the ongoing efforts in the processing, characterization, and consolidation of these materials at ARL. In particular, focus is placed on the methodology used for producing the nanocrystalline metal powders, in both small and large-scale amounts, that are at the center of ongoing research efforts.

This paper provides a brief overview of the ongoing efforts at the Army Research Laboratory on the processing of bulk nanocrystalline metals with an emphasis on the methodologies used for the production of the novel metal powders.

미국 육군 연구소에서 대량 Nanocrystalline 금속 처리

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

큰 면적 기판 기반 Nanofabrication 출장 Dewetting 통해 제어 하 고 사용자 정의 금 나노 입자의

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...