Quantum dot-based, quantitative, and multiplexed assay for tissue staining.
Quantum dot-based, quantitative, and multiplexed assay for tissue staining.
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DOI：
10.1021/am303267g
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发表时间：
2013-04
影响因子：
9.5
通讯作者：
Hong Xu;Jing Xu;Xu Wang;Daqing Wu;Z. Chen;Andrew Wang
中科院分区：
文献类型：
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作者：
Hong Xu;Jing Xu;Xu Wang;Daqing Wu;Z. Chen;Andrew Wang
量子点具有高亮度、高光稳定性、连续吸收和窄发射带宽等优异的光学特性，是开发基于量子点的荧光成像技术的理想光学标记物，可用于福尔马林固定和石蜡包埋（FFPE）组织上的多重癌症生物标志物检测。IHF对于预测患者对癌症化疗或放疗的反应非常重要。基于QD的IHF面临着与用于临床测定的基于有机染料的IHF所遇到的挑战不同的几个挑战。目前的工作涉及其中一些问题。首先，量子点和有机染料的化学稳定性进行了比较。结果表明，QDs在FFPE组织上稳定至少5个月，而有机染料在暴露于光后不久就被光漂白。还研究了各种染色方法。用我公司的一抗（Ab、p16、生存素、EF1α）偶联QD染色的组织上的QD荧光强度与市售方法（分别用一抗p16 Ab和QD标记的二抗羊抗小鼠Ab染色组织）的信号相当。最后，还研究了与QD缀合的Ab的量对组织成像的影响。当Ab：QD比从5增加到30时，组织上的QD荧光信号没有显著增加。此外，测试蛋白G作为衔接蛋白以将Ab连接至QD用于IHF染色。然而，蛋白G在量子点上的适当封闭对于减少串扰是必要的。通过常规Western印迹和免疫组织化学验证基于QD的IHF中的生物标志物定量。本文所包含的结果证明了量子点在生物标志物的多重检测和定量中的有希望的应用。
The excellent optical properties of quantum dots (QDs), such as high brightness, high photostability, continuous absorption, and narrow emission bandwidth, make them ideal as optical labels to develop QD-based immunohistofluorescence (IHF) imaging for multiplexing cancer biomarker detection on formalin-fixed and paraffin-embedded (FFPE) tissues. IHF is very important for the prediction of a patient's response to cancer chemotherapy or radiotherapy. QD-based IHF faces several challenges that differ from those encountered by organic dye based IHF for clinical assays. The current work addresses some of these issues. Initially, the chemical stability of QDs and organic dyes were compared. The results showed that QDs were stable for at least 5 months on FFPE tissue, whereas organic dyes were photobleached shortly after exposure to light. Various staining methods were also studied. QD fluorescence intensity on the tissue stained with primary antibody (Ab, p16, survivin, EF1α) conjugated QDs from our company was comparable to the signal from a commercially available method in which the tissue was stained with a primary p16 Ab and a QD-labeled secondary goat anti mouse Ab respectively. Finally, the effect of the amount of Ab conjugated to QD on tissue imaging was also studied. There was no significant increase in the QD fluorescence signal on tissues when the Ab:QD ratio increased from 5 to 30. In addition, protein G was tested as an adaptor protein to link Ab to QDs for IHF staining. However, the proper blocking of the protein G on QDs was necessary to reduce crosstalk. The biomarker quantification in QD-based IHF was validated by conventional Western blot and immunohistochemistry. The results contained herein demonstrate a promising application of QDs in multiplex detection and quantification of biomarkers.