Name: hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins
Uploaded: 2014-08-07T20:45:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

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><tbody><tr><td>Hydrogel nanoparticles</td><td>Ceres Nanoscience</td><td>CS003</td><td>NanoTrap ESP particles</td></tr><tr><td>18 M&amp;Omega;-cm water</td><td></td><td></td><td>Type 1 reagent grade water</td></tr><tr><td>Tris HCl, 50 mM pH 7.0</td><td>VWR</td><td>IC816116</td><td>50 mM, pH 7</td></tr><tr><td>Acetonitrile</td><td>BDH</td><td>BDH1103-4LP</td><td>Available from VWR</td></tr><tr><td>Ammonium Hydroxide NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>BDH3014</td><td>Available from VWR, assayed at 28-30% NH&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium thiocyanate 25 mM</td><td>Acros Organics</td><td>419675000</td><td>For serum/plasma samples</td></tr><tr><td>Multi-analyte Urine Reagent Strips</td><td>Siemens</td><td>2161</td><td>For urine samples</td></tr><tr><td>Tris-Glycine SDS Sample Buffer (2X)</td><td>Life Technologies</td><td>LC2676</td><td>Use at RT to prevent SDS from precipitating</td></tr><tr><td>Dry bath incubator (100 &lt;sup&gt;o&lt;/sup&gt;C) with heating block</td><td>Barnstead</td><td>11-715-125DQ</td><td>Do not substitute a boiling water bath</td></tr><tr><td>Nitrogen evaporator manifold</td><td>Organomation Associates</td><td>Microvap118</td><td>For serum/plasma samples</td></tr><tr><td>Centrifuge, swing-out rotor</td><td>Sorvall</td><td>Legend series</td><td>50 ml tube capacity, rcf 3,700 x g</td></tr><tr><td>Centrifuge, fixed angle rotor</td><td>Eppendorf</td><td>5424</td><td>1.7 ml microcentrifuge capacity, rcf 16,000 x g</td></tr><tr><td>50 ml conical centrifuge&amp;nbsp;tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>With screw caps for urine samples</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>50-949-755</td><td></td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>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

Research

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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biomarker 디스커버리를위한 Mesoporous 실리카 박막의 낮은 분자량의 단백질 농축

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

연구

생체공학

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

단백질 소화, 한, 및 크기 배제 크로마토그래피 인간의 혈장 및 혈 청에서 Exosomes의 절연을 최적화 하

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

생화학

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

수정 된 침전 법은 요도 엑소 좀을 분리

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

의학

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

단백질 표적 소분자 약물 검사를위한 신속하고 정량적적인 형광 방법

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

전혈에서 고감도 단백질 검출을위한 완전 자동화 된 원심 미세 유체 장치

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

나노 물질에 의해 획득 된 단백질과 대사 산물 코로나특성화를위한 모세관 전기 전구 질량 분광법 접근법

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

화학

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

표적 질량분석법 기반 단백질 분석의 마이크로샘플링 저농도 단백질

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

생물학

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 ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...