친절하게 그림 5의 데이터 수집 및 분석을 지원 마이클 헨리, 더블린 시립 대학은,.이 작품은에 의해 부분적으로 지원되었다 (1) 조지 메이슨 대학 (George Mason University), (2) 이탈리아 / 미국의 틀에서 이탈리아어 IstitutoSuperiore 디 SANITA &#39; 건강의 미국과 복지부, 조지 메이슨 대학 (George Mason University), 그리고 공중 보건의 이탈리아어 자원부, (3) NIH, IMAT 프로그램 보조금 1R21CA137706-01 및 LAL에 1R33CA173359-01, (4) 세레스 Nanosciences, Inc의 협력 계약 .

인간의 혈장과 소변은 조지 메이슨 대학위원회 (Institutional Review Board)가 프로토콜을 승인 한 다음, 동의서와 함께, 건강한 자원 봉사 기증자로부터 수집되었다. 기증자는 동일하게 개별적으로 분석 하였다 25, 42 샘플 세 사이의 백인 남성과 여성 사이에 분포하고 풀링되지 않았다. 혈청 또는 혈장 샘플의 1 나노 입자 처리 플라즈마의 잠재적 낮은 풍부한 바이오 마커는 하이드로 겔 나노 입자 용액에 캡처됩니다. 입자를 세척하고, 원심 분리에 의해 분리 배양 혈장에 첨가하고, 캡처 된 단백질을 용출 하였다. 용출 된 단백질을 질량 분석법 하류 서열 식별 용 질소 기류 하에서 건조시킨다. <ol><li> microcentrifuge 관에서 50 mM의 TrisHCl pH가 7 (500 ㎕의 혈청 + 500 ㎕의 TrisHCl)와 함께 2 : 혈청 1의 500 μl를 희석. </li><li> 500 μL 폴리 (NIPAM / AAC) 핵심 nanop 추가기사. 실온에서 15 분 동안 품어. </li><li> 고정 각 로터가 장착 된 원심 분리기에서 10 분 동안 16,100 XG에 25 ° C를 샘플을 스핀. 제거하고 상층 액을 버린다. </li><li> 펠릿에 500 ㎕의 티오시 안산 나트륨 (25 밀리미터)를 추가합니다. 격렬하게 피펫 팅하여 여러 번 아래 나노 입자를 재현 탁. </li><li> 고정 각도 로터와 원심 분리기에서 10 분 동안 16,100 XG에 25 ° C를 샘플을 스핀. 제거하고 상층 액을 버린다. </li><li> 나노 입자 펠렛을 MilliQ- 물 500 μl를 추가합니다. 격렬하게 피펫 팅하여 여러 번 아래 나노 입자를 재현 탁. </li><li> 고정 각도 로터와 원심 분리기에서 10 분 동안 16,100 XG에 25 ° C를 샘플을 스핀. 제거하고 상층 액을 버린다. </li><li> 신선한 용출 버퍼를 준비 : 깨끗한 튜브에 300 ㎕의 수산화 암모늄에 μL 아세토 니트릴 700를 추가합니다. 주의 : 수산화 암모늄 부식성이다. 적절한 환기와 함께 사용합니다. 주 : 용출 버퍼가 준비이어야ared 즉시 사용하기 전에. 용출 버퍼를 보관하지 마십시오. </li><li> 나노 입자의 펠렛에 용출 버퍼의 300 μl를 추가합니다. 격렬하게 피펫 팅하여 여러 번 아래 나노 입자를 재현 탁. 실온에서 15 분 동안 품어. </li><li> 고정 각도 로터와 원심 분리기에서 10 분 동안 16,100 XG에 25 ° C를 샘플을 스핀. 제거하고 깨끗하고 표시된 microcentrifuge 관에 용출액을 저장합니다. </li><li> 1.10 - 반복 1.9 단계를 반복합니다. 한 microcentrifuge 관에이 용출액을 결합합니다. </li><li> 8 (그림 2 층)로 설정 공기 흐름을, 42.1 ° C에서 질소 증발기 매니 폴드의 질소 흐름에서 용출액을 건조. </li><li> O / N 저장을 위해 실온에서 건조 된 용출액을 저장 또는 장기 저장을 위해 -20 ° C,에서 질량 분석법, 웨스턴 블롯, 또는 ELISA 분석 (옵션) 이전에. </li></ol> 소변 샘플의 2 나노 입자 처리 보통 소변 미만 30 밀리그램 / DL 단백질을 포함하고1 + 혈액보다. 그러나, 많은 질병 / 조건은 소변의 단백질과 혈액의 정상 수준을 변경할 수 있습니다. 소변 샘플에 추가 된 나노 입자의 최적의 양을 결정하는데 원조하기 위해, 나노 입자는 뇨를 수확하기 전에 수행된다. 소변은 생체 소변량에 나노 입자의 비율을 최적화 필요할 수도 극도로 낮은 농도로 존재할 수있다. 이 절차는 다운 스트림 서양 얼룩 분석을위한 소변 샘플의 나노 입자 수확에 대해 설명합니다. <ol><li> 깨끗하고 마른 플라스틱 컵에 소변 샘플을 수집합니다. 22 ㎖의 최소 볼륨이 필요합니다. 분석 할 준비가 될 때까지 보관 소변은 -80 ° C에서 표본. </li><li> RT 또는 4 ° CO / N에서 냉동 소변을 해동. 와류 믹서에 잠깐 섞는다. 50ml의 원뿔형 바닥 폴리 프로필렌 튜브에 소변 중 적어도 22 ㎖에 붓고. </li><li> 15 분 동안 3,700 XG에서 스윙 아웃 로터와 원심 분리기에 소변을 봐. 깨끗한 50 ML 원뿔 바닥 polypr로, 펠렛을 방해하지 않고, 소변을 가만히 따르다opylene 관. 펠렛을 폐기하십시오. </li><li> 다중 분석 &quot;소변 딥 스틱&quot;시약 스트립을 사용하여 소변 검사를 수행합니다. 참고 : 직사광선 및 습도 17, 18에서 멀리 밀폐 된 용기에 시약 스트립을 저장합니다. <ol><li> 깨끗하고 마른 종이 타월에 시약 스트립, 페이스 업을 놓습니다. </li><li> 단계 2.3에서 제조 소변 1 ㎖를 흡인하기 위해 일회용 피펫을 사용합니다. </li><li> 2 분 타이머를 설정하지만, 그것을 시작되지 않습니다. 시약 스트립의 각각의 테스트 패드에 소변이 드랍 - 빠르게 한 분배. 즉시 타이머를 시작합니다. 참고 : 소변 용기에 직접 시약 스트립을 잠수함하지 마십시오. 시약 스트립에 염료 / 화학적 지표 잠재적 소변 용기 내로 시약 스트립의 용출 될 수있다. </li><li> 시약 스트립 용기에 표시된 시간에 대응하는 컬러 코드에 바랭이 개별 시약 스트립의 색을 비교하여 시약 스트립에 대한 다양한 분석 물질의 질적 결과를 기록시약 용기에 TORS. 일반적인 정상적인 소변의 결과는 다음과 같습니다 (일반 또는 음) 혈액, 단백질, 백혈구, 아질산염, 포도당, 케톤, 빌리루빈, 그리고 urobilinogen에 대한; 소변의 pH (5.5-7.0) 비중 (1.001-1.020). 주 : 소변 표본에서 높은 수준의 단백질은 나노 입자 결합 부위에 대한 관심의 단백질과 경쟁 할 수있다. 나노 입자와 단백질을 수확을 극대화하기 위해, 나노 입자 부피 / 소변 체적비가 높은 풍부한 단백질 중 하나에서 제한 경쟁으로 조절 될 수 있거나, 매우 낮은 농도로 존재하는 단백질을 수확하기 위해 최적화 될 수있다. 요 단백 값이 1 + 이상이있는 경우, 아래 단계 2.5에서 나노 입자의 2 배 볼륨을 추가합니다. 주요 피검 물이 매우 낮음 풍부한 단백질 인 경우에는 2 ㎖ (도 3)를 나노 입자의 양을 증가시킬 필요가있을 수있다. </li></ol></li><li> 전송 깨끗한 50 ML 원뿔 바닥 폴리 프로필렌 튜브에 단계 2.3에서 정화 된 소변 20 ㎖. 하지 마십시오소변 튜브의 아래쪽에있을 수있는 어떠한 파편 / 펠렛을 방해. 20 ㎖의 소변 샘플에 나노 입자의 200 μl를 추가합니다. 와류 믹서에 잠깐 섞는다. 요 단백 값이 1 + 이상이면, 20 ㎖의 소변에 나노 입자의 400 μl를 추가합니다. </li><li> 락 / 혼합없이, RT에서 30 분 동안 소변 / 나노 입자 혼합물을 배양한다. </li><li> 10 분 동안 3,700 XG에서 스윙 아웃 로터가 장착 된 원심 분리기에 소변 / 나노 입자 현탁액을 스핀. 주 : 펠릿을 보이는 다음 원심 분리가없는 경우, 부가적인 2 샘플을 스핀 - 7 분. </li><li> 제거하고 상층 액을 버린다. 나노 입자의 펠릿에 500 μL을 MilliQ- 물을 추가합니다. </li><li> 격렬하게 피펫 팅하여 여러 번 아래 나노 입자를 재현 탁. 깨끗한 1.5 ML의 microcentrifuge 관에 나노 입자 용액을 전송합니다. </li><li> 10 분 동안 16,100 XG에 고정 된 앵글 로터를 구비 한 원심 분리기에서 스핀 나노 입자. 주 : 펠릿 FOL 표시가 없으면7 분 - 원심 분리를 lowing의, 추가로 2 샘플을 회전. </li><li> 제거하고 상층 액을 버린다. </li><li> 반복 나노 입자를 씻어 200 μl의 18을 MilliQ- 물 (2 회) 2.8 및 2.11 단계를 반복합니다. </li><li> 신선한 용출 버퍼를 준비 : 깨끗한 튜브에 30 ㎕의 수산화 암모늄에 μL 아세토 니트릴 970를 추가합니다. 주의 : 수산화 암모늄 부식성이다. 적절한 환기와 함께 사용합니다. 참고 : 용출 버퍼가 즉시 사용하기 전에 미리 준비되어 있어야합니다. 용출 버퍼를 보관하지 마십시오. </li><li> 나노 입자의 펠렛에 20 μL 용출 버퍼를 추가합니다. 격렬하게 피펫 팅하여 여러 번 아래 나노 입자를 재현 탁. 실온에서 15 분 동안 품어. </li><li> 10 분 동안 16,100 XG에서 나노 입자 샘플을 스핀. 제거하고 깨끗하고 1.5 ML의 microcentrifuge 관에 뜨는을 저장합니다. 나노 입자 펠렛을 방해하지 마십시오. 생물 학적 쓰레기 펠렛을 폐기하십시오. </li><li> 화학 흄 후드에서 랙에 샘플을 놓습니다. 뚜껑을 열고 inc를30 분 동안 RT에서 ubate. (- 2 시간 1) 또는 건조 할 때까지 40 ° C에서 질소 흐름에서 샘플을 배치. </li><li> 샘플 15 ㎕의 트리스 - 글리신 SDS 샘플 버퍼 (2 배)를 추가합니다. (100)에서 열 열거 나 튜브에 남아있는 볼륨까지 모자와 5 분 동안 건조 열 블록 ° C는 더 이상 20 μL 없습니다. 참고 : 끓는 물에 목욕을 사용하지 마십시오. 수욕에서 습도는 버퍼의 증발을 방지한다. </li><li> 튜브에 뚜껑을 놓습니다. 열 블록에서 튜브를 제거합니다. 샘플은 -80 ° C에서 저장 또는 다운 스트림 서양 얼룩 분석을 위해 즉시 사용할 수 있습니다. </li></ol>

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Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

임상 관련성 혈청 또는 혈장 시료 전체적으로 유기체의 상태에 관한 정보의 다양한 소스를 제공 할 수있는 단백질 및 펩티드를 순환 미량을 포함하는 것으로 생각된다. 혈청 단백질 체학의 약속에도 불구하고, 임상 적 이익을 10, 11, 16, 25로 바이오 마커의 발견과 번역을 훼방 세 가지 기본 심각한 생리적 장벽이있다. 1 중요한 진단 ?...

인간 게놈 시퀀싱의 완성에도 불구하고 상당한 진전은 초기 질환의 예측 식별 생체 제되지 않았거나 그 치료 결과, 1 또는 예후와 상관 관계. 진행의 부족이 이유 중 하나는 많은 중요한 잠재적 바이오 마커는 종래의 질량 분석 등의 바이오 마커 발견 플랫폼의 검출 한계 이하의 농도로 존재한다는 것이다. 질량 분석법 (MS)과 다중 반응 모니터링 (MRM)을 전형적으로 검출 감도가 50 이상 / ㎖ 겨 동안 50 PG / ㎖ 및 10 NG / ㎖ 사이의 범위에있는 임상 화학 가을 면역 분석법에 의해 측정 된 분석 물의 대부분 . 특히 질병의 초기 단계에서 많은 바이오 마커는, 종래와 MRM MS (2)에 의해 검출 될 수 없다는 것을 의미한다. 또한 이러한 복잡한 생물학적 유체에서 알부민 및 글로불린 같은 고 풍부 단백질의 존재는 종종 억 배 EXC 의해 마스크ESS 미량, 저 분자량의 단백질 및 펩티드 3, 4. 따라서 여러 샘플 준비 작업 단계가 질량 분석 시퀀싱 및 식별에 앞서 요구된다. 하나는 이러한 준비 단계는 상업적으로 이용 가능한 고갈 열 5-8로 높은 풍부한 단백질의 고갈을 고용하고있다. 그들은 종종 비공유 제거되는 캐리어 단백질과 연관되어 있기 때문에 불행하게도이 공정은 바이오 마커 후보의 수율의 감소를 이끈다. 샘플이 수집되면 또 다른 문제는 바이오 마커 후보의 전 생체을의 안정성으로 표시됩니다. 단백질은 내인성 또는 외인성 단백질 분해 효소 (9)에 의해 분해 될 수 있습니다. 10-13 열화로부터 단백질을 보호하는 동시에 하이드로 겔 나노 입자, 분석의 범위 내의 수준으로 바이오 마커 추정 농도를 증폭함으로써 이러한 중요한 과제를 초월 할 수있다. 그것은 일을 주목해야합니다LMW에서 혈액 내 단백질은 작은 그대로 단백질뿐만 아니라 큰 단백질 단편의 혼합물이다. 60 kDa의보다 큰 조직 유래 단백질은 수동적으로 혈관 기저막을 통해 혈류에 들어가기에 너무 클 수 있지만 그것들은 펩티드 또는 단백질의 단편 (14)과 같은 혈액에서 표현 될 수있다. 우리의 목표는 질병의 조기 진단, 치료에 대한 환자 층화하고, 치료에 대한 반응을 모니터링하기위한 후보가 될 수있는 신규 한 생체 순환을 측정하는 방법이다. 우리의 나노 입자를 동시에 작은 단백질과 펩타이드를 캡처하고 시작 볼륨에 따라 100 배에 그들을 집중하면서 선택적으로, 높은 풍부한 면역 글로불린과 알부민을 제외 만들어집니다. 우리 그룹이 성공적으로 단백질 및 펩타이드에 대해 높은 친 화성 분자 미끼 작용할 수 작은 유기 염료의 세트를 식별. 결합 단백질 염색으로 인해 소수성 및 정전 기적 상호 작용의 조합에 의한 것으로 생각된다의. 염료에 방향족 고리는 단백질의 소수성면 (11)에 포켓을 통해 단백질 인터리빙. 미끼는 자신의 화학에 따라 분석의 선택된 클래스에 대한 특별한 친화력을 보여줍니다. 미끼는 단백질 또는 펩티드, 알부민과 같은 담체 단백질과 경쟁. 저 분자량 단백질 / 펩타이드는 입자에 포획된다. 알부민과 글로불린과 같은 고 분자량의 단백질 때문에 하이드로 겔 (11) (도 1)의 제한으로 인해 기공 체질 능력 입자 들어가는 것이 방지된다. 하이드로 겔 나노 입자 (11) 황산 암모늄 침전에 의해 개시 중합에 의해 합성된다. N-이소 프로필 아크릴 아미드 (NIPAM)은 아크릴산 (AAC) 및 알릴 아민 (AA)와 가교제 N, N&#39;-메틸렌 비스 (BIS)의 코 모노머는 희석 조건 (1)에서 6 시간 동안 70 ° C에서 반응하도록 허용1, 13. 폴리 높은 단백질 결합 친화도 (N-이소 프로필 아크릴 아미드 - 코 - 아크릴산) 공유 (아미노 - 함유 염료를 통합 즉., sulfonatedanthraquinonetriazine 염료)를 나노 입자로를 통해 달성 (폴리 (NIPAM-CO-AAC) nanoparticlesis 염료의 친수성 / 소수성 특성에 따라 수용성 또는 유기 용매에서 수행 아미드 화 반응은 11 anthraquinonetriazine 염료의 염소 원자를 가진 나노 입자의 아민 기의 13. 친 핵성 치환은 염료 - 함유 폴리 (NIPAM를 만드는 데에 이용된다 -CO-알릴 아민) (AA)는, 나노 입자 (11) 12. 두 단계의 중합 방법은 비닐 술폰산 (VSA) (11), (13)의 외부 쉘 나노 입자를 함유하는 하이드로 겔을 생성하는 데에 이용된다. 하이드로 겔 나노 입자는 전체 혈액, 혈장, 혈청, 뇌척수액, 땀, 뇨 등의 다양한 생물학적 유체에 적용될 수있다. 한 단계에서, 용액, Nanoparticles는 (분 이내) 신속한 격리 및 저 분자량의 농도는 10, 11, 13, 15-18 수행 분석 물. 단백질이 후속하여 나노 입자로부터 용출 검출 웨스턴 분석법 10, 11, 13, 15, 18, ​​22 질량 19-21 블롯, 23, 면역 / ELISA 10, 11, 15, 18, ​​또는 역상 단백질 마이크로 어레이 (16), 24 분석. 나노 입자는 화학 미끼로 작용하고, 코어 또는 코어 쉘 구조, 캡처를 제시하고 미끼 / 쉘 물리 화학적 특성에 따라 단백질을 집중한다. 나노 입자에 혼입 다른 염료 따라서 염료 선호도에 기초하여 효율, 용액의 pH, 및 고 단백질 풍부 (13)를 경쟁의 존재 / 부재를 가변으로 단백질의 상이한 서브 세트를 캡처한다. 또한, 용액의 부피에 관련하여 나노 입자의 양은 나노 입자에서 단백질 수율에 영향을 미칠 것이다. 이러한 측면하이드로 겔 나노 수확 단백질을 다량 함유 혈장 샘플로부터 수확하는 세 가지 단백질 나노 입자를 사용하여 입증 베이트, 전형적으로 많은 양의 단백질을 포함하지 않는 소변 샘플에서된다. 이 프로토콜에서는 수확 및 폴리 (NIPAM-CO-AAC), 폴리 (NIPAM / 염료), 및 코어 - 쉘 나노 입자를 사용하여 혈장 샘플에서 종양 괴사 인자 알파 (TNFα)를 집중을 설명 (폴리 (NIPAM-CO-VSA)) . 폴리 (NIPAM / 염료) 나노 입자는 결핵균에 감염된 개인을 모방하기 위해 인간의 소변 샘플에 추가 된 마이 코박 테 리움 종의 항원을 집중 표시됩니다.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

새로운 바이오 마커 발견에 더 민감하고 특정 질환 검출을 제공하는데 결정적인 역할을한다. 생물학적 유체에 존재 불행히도 많은 미량 생체 용이 질량 분석법 또는 그들이 매우 낮은 농도로 존재 불안정성이며, 종종 알부민 또는 면역 글로불린과 같은 높은 풍부한 단백질에 의해 마스킹되기 때문에 면역 분석법으로 검출 될 수 없다. 미끼 함유 폴리 (N-이소 프로필 아크릴 아미드)는 (NIPAM) 기반 나노 입자는 이러한 생리적 장벽을 극복 할 수있다. 한 단계에서 그들은 캡처 집중 체액에서 바이오 마커를 보존 할 수 있습니다. 저 분자량 분석은 나노 입자의 코어를 선택하고 높은 친 화성 단백질 베이트 행동 다른 유기 화학 염료에 의해 포착된다. 나노 입자는 몇 배에 의해 관심의 단백질을 집중 할 수 있습니다. 이 집중 계수는 단백질 내에되도록 단백질 수준을 증가시키기에 충분한현재 질량 분석기, 웨스턴 블롯 및 면역 분석법의 검출 한계. 나노 입자는 생체 체액의 과다와 함께 배양 할 수 있고, 그들은 알부민 및 기타 고 분자량 단백질을 제외하고 크게하면서 저 분자량의 단백질 및 펩티드의 농도로 농축 할 수있다. 우리의 데이타는 특정 분석 물질의 농도가 10,000 배 증폭 이전에 탐지 바이오 마커를 검출하는 질량 분석 및 면역 분석을 가능하게 달성 될 수 있음을 보여준다.

하이드로 겔 나노 입자 크기와 균일 폴리 (NIPAM-AAC) 입자 사이의 일괄 처리 내에서 매우 높은 수율과 재현성으로 제작되었다. 입자는 캡처, 저장, 및 단백질의 용출 (적어도 48 시간)에 필요한 시간 동안 RT에서 매우 좋은 콜로이드 안정성을 가지고 있고, 나노 입자가 석출 (도 1) (11)이 관찰되지 않았다. 콜로이드 안정성은 나노 입자에 의한 신속...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

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

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 ...

hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

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JoVE Journal

Bioengineering

13.8K 조회수.  George Mason University. 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.

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

세포 네트워크의 창조를위한 플라즈마 리소그래피 표면 Patterning

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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생체공학

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

설계 및 인공 세포 외 기질의 건설 (aECM) 단백질에서<em&gt; 대장균</em&gt; 피부 조직 공학

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

섬세한 조직 조작을위한 사용자 정의 소프트 로봇 공압 그리퍼 장치의로드 기반의 제조

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

액정 나노 입자 캐리어의 소수성화물 캡슐화의 대상 플라즈마 멤브레인 배달

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

생리 활성 단백질 또는 펩 티 드 히드로 광화학을 사용 하 여 생물 학적 응용에 대 한에 패턴화

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

조직 Microcolumns 수확 용

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

날조는 신장 피 질 세포 외 매트릭스 파생 하이드로 겔

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

박테리아 스크리닝, 선택 및 격리를 위한 광분해성 하이드로겔 인터페이스

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

강력한 접착력 하이드로겔, 젤라틴 O-니트로소벤즈알데히드의 합성

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...