Roda A, Pasini P, Musiani M, Girotti S, Baraldini M, Carrea G, Suozzi A
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Anal Chem. 1996 Apr 1;68(7):1073-80. doi: 10.1021/ac951062o.
The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 μm. Other applications, such as the alkaline phosphatase localization in 8 μm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 μm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.
在宏观层面并结合光学显微镜，从灵敏度、空间分辨率、准确性、精密度和样品几何形状等方面评估了用于定量发光分析的低光成像发光仪的分析性能。该系统分别使用1,2-二氧杂环丁烷和鲁米诺/对碘苯酚或吖啶羧酸酯作为化学发光底物，能够检测到400 amol的碱性磷酸酶和辣根过氧化物酶等酶。研究了尼龙网固定化碱性磷酸酶的酶活性和空间分布；该系统能够以低至1μm的空间分辨率对固定化酶进行定量。还研究并优化了其他应用，如在8μm肠黏膜冰冻切片中碱性磷酸酶的定位、定量免疫细胞化学和斑点印迹DNA杂交反应。该系统还用于感染人成纤维细胞中巨细胞病毒DNA的原位杂交检测。使用该酶的化学发光底物，通过地高辛配体标记的探针和碱性磷酸酶标记的抗地高辛配体抗体揭示病毒基因组的存在。发光信号强烈且稳定，并且能够在单细胞内对探针进行成像和定量，细胞核中的强度更高，空间分辨率低至1μm，背景非常低。结果表明，该技术是一种超灵敏且强大的分析工具，可在微观水平上定位和定量生物分子，适用于许多生物分析应用。
