We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.

참고 : 분자 생물학 수준의 물이 모든 버퍼 및 솔루션 준비에 사용됩니다. 모든 일회용 튜브 (마이크로 원심과 PCR) 및 피펫 팁 DNase-과의 RNase-무료입니다. 사용하기 전에 모든 화학 물질에 대한 물질 안전 보건 자료 (MSDS)를 참조하십시오. 적절한 개인 보호 장비 (PPE)를 사용하는 것이 좋습니다. 앱타 머는 재고 솔루션 1. 준비 <ol><li> 상업적으로 polydeoxynucleotide의 2 가닥을 구입합니다. 고성능 액체 크로마토 그래피 (HPLC)로 정제와 두 가닥을 주문한다. 1 (GD-앱 타머를) 가닥 : 5 &#39;- / 56 FAM / AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; 스트랜드 2 (QS) : 5&#39;-GTCCCGAGAGCCT / 3Dab / -3 &#39; </li><li> 하나님 - 앱 타머 및 QS 100 μM 각각의 원액을 물에 각각의 가닥을 녹인다. </li><li> -20 ° C에서 이러한 솔루션을 저장합니다. 솔루션은 지금까지 3 년 동안 안정하다. </li><li> FREEZ을 최소화하기 위해전자 해동 사이클, 10 μL 씩의 주식 솔루션을 저장합니다. </li></ol> 2 배속 하나님 센서 솔루션의 2. 준비 <ol><li> 상기 분석 버퍼를 준비한다 (20 mM의 HEPES, 2 밀리미터의 MgCl 2, 150 mM의 염화나트륨, 5 mM의 KCl을). 수산화 나트륨과 염산으로 pH를에 ~ 7.4를 조정하고 0.2 μm의 PES 막으로 멸균 일회용 병 톱 필터를 통해 필터링합니다. 멸균 병에 보관합니다. 여과하고 (실온에서) 제대로 저장하는 경우, 버퍼는 지금까지 2 년 동안 안정하다. </li><li> 분석 버퍼 497 μL와 (단계 1.2)에 QS 원액의 2 μL (단계 1.2)에 하나님 - 앱 타머 주식 솔루션의 1 μL를 희석. 소용돌이를 사용하여 잘 섞는다. 배속 GD-센서 용액에서의 농도는 각각 200, 400㎚이다. 주 - 검량선 및 조영제 솔루션 3+ 하나님 7이 다양한 농도에서 제조 한 배 GD-센서 용액의 부피는 6을 테스트하기에 충분한 500 μL 인(단계 3). 각 샘플은 384 웰 플레이트에 중복 우물을 줄 것이다. <ol><li> 시험해야 하나님 3+ 솔루션의 수에 따라 2 배 GD-센서 용액의 양을 조정한다. </li></ol></li><li> 각 튜브에 50 μL로, 9 PCR 튜브에 2 배 하나님 센서 솔루션을 전송합니다. 열 자전거 타는 사람에 튜브를 놓습니다. </li><li> 95 ° C로 튜브 용액을 가열하는 열 자전거 타는 사람에 프로그램을 설정, 5 분 동안 유지 한 후 천천히의 속도로 (15 분 ~ 동안 25 ° C에 솔루션을 냉각 ~ 0.05-0.1 ° C / 에스). 가열 및 냉각 사이클은 하나님-앱 타머와 QS 사이의 최적의 하이브리드을 보장하는 것입니다. 불완전 담금질하고 센서의 높은 배경 형광 부분 혼성화 결과. 열 사이 클러를 사용할 수없는 경우, 대신에 뜨거운 물이 욕을 사용하여이 과정을 수행한다. </li><li> 일단 25 ℃까지 냉각하고, 즉시 용액을 사용하거나 할 준비가 될 때까지 (약 2 시간까지) 열 사이 클러에 보관익숙한. 수욕은 가열에 사용하는 경우에는 물을 서서히 실온으로 냉각대로 용기에 튜브를 떠난다. </li></ol> 3. 형광 교정 곡선을 구축하고 하나님 대비 에이전트의 솔루션에 3+ Unchelated 하나님의 존재를 감지 <ol><li> 상기 분석 버퍼 고체 GdCl 3 (단계 2.1에서와 같은 버퍼)에 녹인다. </li><li> 시리얼 희석 통해 검량선 (2 × 용액)에 대해 원하는 최종 농도의 100 배에 μL 마이크로 원심 튜브 6 다른 하나님 3+ 용액을 각각 준비한다. <ol><li> 예를 들어, 0에 대한 보정을 구성, 50, 100, 200, 400 (NO GdCl 3 만 버퍼링)하고 하나님 3+ 800 nM의 0, 100, 200, 400, 800 및 1600 nM의 함유 용액을 제조 이온의. 확인은 음성 대조군 0 nM의 하나님 3+와 &#39;빈&#39;을 항상 포함 할 수 있습니다. </li></ol></li><li> 시험 될 조영제 녹이고분석 버퍼에 에드. 일련의 희석을 통해 조영제 용액의 2 또는 3 개의 농도를 준비한다. 참고 : 조영제 솔루션의 3 가지 농도를 테스트하는 것이 좋습니다. 이것은 이러한 농도는 선형 범위 내에 있도록한다. 선형 관계를 표시하지 않은 시험 샘플은, 조영제의 농도를 감소하는 경우 사용된다. </li><li> 열 자전거 타는 사람 중 단계 2.5에서 2 배 하나님 센서 솔루션을 포함하는 PCR 튜브를 타고. </li><li> 2 배속 하나님 센서 솔루션을 포함하는 9 PCR 튜브 (6)에 단계 3.2에서 각각 하나님 3+ 용액 50 μL를 추가합니다. 로 pipetting 아래로 섞는다. 각 PCR 튜브는 이제 원하는 하나님 3+의 농도를 테스트 할, 100 nm의 하나님 - 앱 타머, 200 nm의 QS가 포함되어 있습니다. </li><li> 배속 GD-센서 함유 용액 나머지 PCR 튜브에 단계 3.3에서 조영제 용액 50 μL를 추가한다. 최대 피펫 팅에 의해 몇 번 아래로 잘 섞는다. </li><li> 의 S를 품어실온에서 약 5 분 동안 PCR 튜브에 olutions. 그들은 최대 30 분 동안 방치 될 수있다. </li><li> 384 웰 플레이트에 각 튜브의 45 μL를 전송합니다. 각 PCR 튜브 중복 우물을 줄 것이다. </li><li> 접시 리더에 각 웰의 형광을 기록합니다. 공급 업체의 웹 사이트에 나와있는대로 하나님 센서 설계에 사용되는 형광 물질 (FAM)는 각각 495과 520 나노 미터의 여기 및 방출 최대 값을 갖는다. 판 독자가 monochromator- 또는 필터 기반인지에 따라 적합한 여기 및 방출 파장 또는 필터를 선택합니다. </li><li> 하나님 3+의 농도에 대한 임의의 형광 단위 (AFU)에서 형광의 그래프를 플롯. </li><li> 하나님 3+의 농도에 대한 형광 배 변화와 그래프를 플롯. (하나님의 영 nM의 3 이상 포함) &#39;빈&#39;솔루션의 AFU 각 농도의 AFU을 나누어 형광 배의 변화를 계산한다. 형광 배의 변화는 N을 허용합니다결과 ormalization의 AFU 표시 일부주기 (다른 일 등)가 유사해야한다. </li><li> 이 0 ㎚ GdCl 3 (단 버퍼)를 함유하는 용액 인 조영제를 함유하는 용액과 &quot;빈&quot;의 형광 방출을 비교한다. 주 : GBCA 용액 높은 형광 조영제의 추가의 정제를 필요로 할 수있다 unchelated 하나님 3+의 존재를 의미한다. unchelated 하나님 3+의 양은 단계 3.10 3.11 구성된 검량선을 이용하여 추정 될 수있다. </li></ol>

<ol>
	<li>Shen, C., New, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promising+strategies+for+Gd-based+responsive+magnetic+resonance+imaging+contrast+agents.">Promising strategies for Gd-based responsive magnetic resonance imaging contrast agents.</a> Curr. Opin. Chem. Biol. 17 (2), 158-166 (2013).</li><li>Cheong, B. Y. C., Muthupillai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrogenic+systemic+fibrosis:+a+concise+review+for+cardiologists.">Nephrogenic systemic fibrosis: a concise review for cardiologists.</a> Tex. Heart Inst. J. 37 (5), 508-515 (2010).</li><li>Hao, D., Ai, T., Goerner, F., Hu, X., Runge, V. M., Tweedle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+contrast+agents:+basic+chemistry+and+safety.">MRI contrast agents: basic chemistry and safety.</a> J Magn. Reson. Imaging. 36 (5), 1060-1071 (2012).</li><li>Telgmann, L., Sperling, M., Karst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+gadolinium-based+MRI+contrast+agents+in+biological+and+environmental+samples:+a+review.">Determination of gadolinium-based MRI contrast agents in biological and environmental samples: a review.</a> Anal. Chim. Acta. 764, 1-16 (2013).</li><li>Frenzel, T., Lengsfeld, P., Schirmer, H., H&#252;tter, J., Weinmann, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+gadolinium-based+magnetic+resonance+imaging+contrast+agents+in+human+serum+at+37+degrees+C.">Stability of gadolinium-based magnetic resonance imaging contrast agents in human serum at 37 degrees C.</a> Invest. Radiol. 43 (12), 817-828 (2008).</li><li>Loreti, V., Bettmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+MRI+contrast+agent+Gd-DTPA+by+SEC-ICP-MS.">Determination of the MRI contrast agent Gd-DTPA by SEC-ICP-MS.</a> Anal. Bioanal. Chem. 379 (7), 1050-1054 (2004).</li><li>Telgmann, L., 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=Speciation+and+isotope+dilution+analysis+of+gadolinium-based+contrast+agents+in+wastewater.">Speciation and isotope dilution analysis of gadolinium-based contrast agents in wastewater.</a> Environ. Sci. Technol. 46 (21), 11929-11936 (2012).</li><li>Cleveland, 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=Chromatographic+methods+for+the+quantification+of+free+and+chelated+gadolinium+species+in+MRI+contrast+agent+formulations.">Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations.</a> Anal. Bioanal. Chem. 398 (7), 2987-2995 (2010).</li><li>Edogun, O., Nguyen, N. H., Halim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+single-stranded+DNA-based+assay+for+detecting+unchelated+gadolinium(III)+ions+in+aqueous+solution.">Fluorescent single-stranded DNA-based assay for detecting unchelated gadolinium(III) ions in aqueous solution.</a> Anal. Bioanal. Chem. 408 (15), 4121-4131 (2016).</li><li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1 (5), 184-188 (2006).</li><li>Johansson, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+reporter-quencher+pairs+for+efficient+quenching+through+formation+of+intramolecular+dimers.">Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers.</a> Methods Mol. Biol. 335, 17-29 (2006).</li><li>Sherry, A. D., Caravan, P., Lenkinski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+gadolinium+chemistry.">A primer on gadolinium chemistry.</a> J. Magn. Reson. Imaging. 30 (6), 1240-1248 (2009).</li><li>Shakhverdov, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-relaxation+mechanism+of+fluorescence+quenching+in+complexes+of+lanthanide+ions+with+organic+ligands.">A cross-relaxation mechanism of fluorescence quenching in complexes of lanthanide ions with organic ligands.</a> Opt. Spectrosc. 95 (4), 571-580 (2003).</li><li>Brittain, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicrogram+determination+of+lanthanides+through+quenching+of+calcein+blue+fluorescence.">Submicrogram determination of lanthanides through quenching of calcein blue fluorescence.</a> Anal. Chem. 59 (8), 1122-1125 (1987).</li></ol>

The authors declare that they have no competing financial interests.

앱타 머는 기반 GD-센서 관측 unchelated 하나님 3+의 농도에 비례하는 형광 방출의 증가 사용. 사용한 시료의 양을 최소화하기 위해, 분석은 웰 당 45 μL의 총 샘플 부피의 384 웰 마이크로 플레이트에서 실행될 수있다. 이 설계에서, 플루오 레세 인 (FAM) 및 dabcyl DAB ()의 선택은 주로 시약의 비용에 기초 하였다 발광 파장, 형광의 다른 쌍을 수정 및 소광 제 (11)를 사용할 수있다. <p class...

기술의 고유의 감도에 의해 제한된다 임상 진단에서 자기 공명 영상 (MRI)의 중요성이 증가는 신규 가돌리늄 계 조영제 (GBCAs 1)의 개발에 대한 연구의 빠른 성장을 가져왔다. GBCAs은 이미지 품질을 개선하기 위해 투여되는 분자이며, 그들은 일반적으로 여러 자리 리간드에 배위 3가 가돌리늄 이온 (하나님 3+)의 화학 구조를 갖는다. 이 복합체는 독성 3+ unchelated 하나님으로 매우 중요합니다; 이는 신장 질환 또는 장애이 일부 환자에서 신원 성 전신 섬유증의 발병과 연관되어있다. 따라서, 수성 자유 이온을 검출하는 GBCAs의 안전을 확보하는 수단이있다. GBCA 솔루션 unchelated 하나님 3+의 존재는 종종 리간드 및 이온 복합체의 해리 또는 displacemen 간의 불완전한 반응의 결과다른 생물학적 금속 양이온 (3)에 의한 t. 현재 하나님 3+, 융통성 및 적응성 (4)의 측면에서 크로마토 그래피 및 / 또는 분광 최고 순위를 의존들의 존재를 결정하기 위해 사용하는 여러 기술 중. 강점 중 높은 감도 및 정확도 (리스트를, 복수 하나님 3+ 복합체의 동시 정량 (인간 혈청 5 소변 헤어 6 폐수 7 및 조영제 제제 8 등) 다양한 시료 매트릭스를 분석하는 능력이다 연구의 2013 년 이전은 Telgmann 등.) (4)에 의해 종합적인 검토에 설명되어 있습니다. 유일한 단점은 이들 방법 중 몇몇은 몇몇 실험실에 액세스 할 수있다 (예 : 유도 결합 플라즈마 질량 분석)을 측정 장치 (4)를 필요로한다는 것이다. 연구 및 개념 증명 수준 AR에 참신한 GBCA 검색의 맥락elatively 더욱 편리하고 신속하고 비용 효율적인 분광 기반 방법 (예 : 비스 - UV 흡수 또는 형광 등) 유용한 대안이 될 수있다. 마음에 이러한 응용 프로그램으로, 수성 하나님 3+에 대한 형광 앱 타머 기반 센서 (9)을 개발 하였다. 앱 타머 (GD-앱 타머)는 지수 농축 (SELEX) (9)에 의한 리간드의 체계적인 진화의 과정을 통해 분리하여 염기의 특정 순서와 44 염기 길이의 단일 가닥 DNA 분자이다. 형광 센서에 앱 타머에 적응하기 위해, 형광 물질은 13 상보적인 염기 (도 1)를 통해 급냉 스트랜드 (QS)와 혼성화되는 가닥의 5 &#39;말단에 부착된다. QS는 3 &#39;말단에 어두운 소광 분자 태그입니다. 1 이루어지는 하나님 3+ 센서 (GD-센서)의 부재에있어서 : 각각 GD-앱 타머 및 QS 2 몰비 최소 형광 방출 인해 t있을 것이다소광에 형광에서 오 에너지 전달. 수성 하나님 3+의 첨가는 형광 방출 증가의 결과로, GD-앱 타머에서 QS를 대체 할 것이다. <img alt="그림 1" src="/files/ftp_upload/55216/55216fig1.jpg" /> 형광 (형광)과 dabcyl 태그로 13베이스 긴 담금질 가닥 (QS) 태그로 44베이스 긴 앱 타머 (GD-앱 타머) (어두운 소광)로 구성되어 그림 1. 센서 (하나님 센서) . unchelated 하나님 3+의 부재에서, 센서의 형광이 최소이다. 하나님 3+의 첨가에 의해, QS의 변위가 발생하고, 형광 발광의 증가가 관찰된다. <a href="http://ecsource.jove.com/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 현재 존재 한 일반적으로 사용되는 광학적 기반 방법에 대한 검출수성 하나님의 3 이상을 보내고. 이 분석은 이온 10 킬에 따라 433 nm의 573에서 최대 흡수 파장의 변화를 겪는다 분자 크 실레 놀 오렌지를 사용합니다. 이들 두 흡수 최대 값의 비율은 unchelated 하나님 3+의 양을 정량화 할 수있다. 두 가지 방법은, 타겟 선택도, 정량 분석의 선형 범위 및 검출 양상 (예 : pH 및 사용되는 버퍼 용액의 조성과 같은) 상이한 반응 조건을 가지고, 상기 앱 타머 센서는 크 실레 놀 오렌지 분석에 대한 대안 (또는 보완 될 수있다)는 9.

<table><tbody><tr><td>Gd-aptamer</td><td>IDTDNA</td><td>Input sequence and fluorophore modification in the order form</td><td>A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Quenching strand</td><td>IDTDNA</td><td>Input sequence and quencher modification in the order form</td><td>A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Molecular biology grade water</td><td></td><td></td><td>No specific manufacturer, both DEPC or non-DEPC treated work equally well</td></tr><tr><td>Gadolinium(III) chloride anhydrous</td><td>Strem</td><td>936416</td><td>Toxic</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Magnesium chloride anhydrous</td><td>MP Biomedicals</td><td>0520984480 - 100 g</td><td></td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P333-500</td><td></td></tr><tr><td>Sodium hydroxide, pellets</td><td>Fisher Scientific</td><td>BP359</td><td>Corrosive</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>SA49</td><td>Toxic and corrosive</td></tr><tr><td>384-well low flange black flat bottom polystyrene NBS plates</td><td>Corning</td><td>3575</td><td>Plates which are suitable for fluorescence reading are required.</td></tr><tr><td>Nalgene Rapid-Flow sterile disposable bottle top filter</td><td>Thermo Scientific</td><td>5680020</td><td>The bottle top is fitted with 0.2 micron PES membrane</td></tr><tr><td>Disposable sterile bottles 250 mL</td><td>Corning</td><td>430281</td><td>A larger or smaller bottle may be used</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>Micropipets</td><td></td><td></td><td>No specific manufacturer</td></tr><tr><td>Pipet tips (non filter) of appropriate sizes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek Synergy H1</td><td></td><td>Plate readers from other manufacturers would work equally well</td></tr></tbody></table>

an aptamer-based sensor for unchelated gadolinium(iii)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

unchelated 하나님 3+의 존재 GD-센서 용액의 전형적인 형광 변화는도 2에 도시되어있다. 방출은 형광 배 변화 (그림 2A) 또는 임의의 단위로 읽기 원시 형광 (그림 2B)로 나타내 어질 수 (AFU). 두 플롯&gt; 3 μM에서 신호의 1 μM 및 채도 아래 하나님의 농도에 대한 선형 범위 3+와 매우 유사 교정 곡선을 얻었다. 검출 한계는 3 신?...

Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

Unchelated 가돌리늄위한 앱 타머 기반 센서 (III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

an-aptamer-based-sensor-for-unchelated-gadolinium-iii

Research

JoVE Journal

Chemistry

An Aptamer-based Sensor for Unchelated Gadolinium(III)

7.2K 조회수.  California State University, East Bay. The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor. 

비디오: An Aptamer-based Sensor for Unchelated GadoliniumIII

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

EOB-DTPA와의 조지아 (III) 단지에 대한 조사 그것<sup&gt; (68)</sup&gt; 조지아 방사성 표지 된 아날로그

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

연구

화학

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

준비 및<em&gt; 체외</em&gt; 자기 공명 영상에 대한 덴드리머 기반 대조 대리인의 특성

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

작은 분자 CLT1 펩타이드 표적 조영제와 전립선 암의 MR 분자 영상

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

의학

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

멀티 모달 분자 이미징 애플리케이션을위한 Biofunctionalized 프 러시안 블루 나노 입자

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

생체공학

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

자기 공명 이미지 지침에 열성 리포좀에서 시스플라틴의 트리거 된 릴리스를위한 맞춤 설계 레이저 기반의 가열 장치

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

질병 진단을위한 휴대용 이미징 및 분석과 결합 자기 부상

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

<meta charset="utf8"></head><body>새로운 철 킬레이트 검출을 위한 형광 분석

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

<meta charset="utf8"></head><body>저자 스포트라이트: 새로운 철 킬레이트가 암세포 대사에 미치는 영향 평가

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

철을 킬레이트화하는 새로운 화합물의 능력을 확인하기 위한 정량화 가능하고 저렴한 cell-free 형광 방법 

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

암 연구학

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Unchelated 가돌리늄위한 앱 타머 기반 센서 (III)

요약

더 많은 비디오 탐색

이 비디오의 챕터

EOB-DTPA와의 조지아 (III) 단지에 대한 조사 그것

준비 및

작은 분자 CLT1 펩타이드 표적 조영제와 전립선 암의 MR 분자 영상

멀티 모달 분자 이미징 애플리케이션을위한 Biofunctionalized 프 러시안 블루 나노 입자

자기 공명 이미지 지침에 열성 리포좀에서 시스플라틴의 트리거 된 릴리스를위한 맞춤 설계 레이저 기반의 가열 장치

질병 진단을위한 휴대용 이미징 및 분석과 결합 자기 부상

철을 킬레이트화하는 새로운 화합물의 능력을 확인하기 위한 정량화 가능하고 저렴한 cell-free 형광 방법

철을 킬레이트화하는 새로운 화합물의 능력을 확인하기 위한 정량화 가능하고 저렴한 cell-free 형광 방법

철을 킬레이트화하는 새로운 화합물의 능력을 확인하기 위한 정량화 가능하고 저렴한 cell-free 형광 방법

Automated Preparation of [⁶⁸Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment