Roda A, Pasini P, Baraldini M, Musiani M, Gentilomi G, Robert C
Department of Pharmaceutical Sciences, University of Bologna, Italy.
Anal Biochem. 1998 Mar 1;257(1):53-62. doi: 10.1006/abio.1997.2514.
A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-micron-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2 and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens and in situ hybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2 system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.
已开发出一种化学发光系统，用于超灵敏定量分析以及生物分子（如抗原、酶、抗体、组织或细胞中的DNA探针）空间分布的可视化。该系统由连接到光学显微镜的低光成像光导摄像管摄像机组成，能够在单光子水平测量光，并对分析物的亚细胞分布进行三维图像分析。使用适当的化学发光底物可以测定酶或酶标记的生物特异性试剂的浓度和空间分布。分析物也可以通过终止于发光的偶联酶促反应来测定。固定有辣根过氧化物酶的环氧乙烷丙烯酸珠（直径250微米的大孔颗粒）已被用作模型系统，以根据不同化学发光底物（如鲁米诺/增强剂/H₂O₂和吖啶羧酸酯/H₂O₂）的信号强度和空间分辨率来优化实验条件。固定有乙酰胆碱酯酶的环氧乙烷珠的定位也已用于优化一个系统，其中初级酶的检测和定位涉及溶液中的两种二级酶，胆碱氧化酶和辣根过氧化物酶，最终导致发光。使用过氧化物酶标记的抗体或cDNA探针在细胞中进行了检测病毒抗原的免疫酶反应和检测病毒DNA（巨细胞病毒、单纯疱疹病毒）的原位杂交试验，并评估了不同化学发光底物对该酶的分析性能。获得的结果表明，有可能在单细胞中清晰地成像生物探针，并且当鲁米诺/H₂O₂系统与增强剂结合使用时，如在ECL和SuperSignal Ultra试剂中，过氧化物酶是一种合适的标记；其他底物，如Lumigen PS-3，尽管具有足够的可检测性，但由于激发发射物种的半衰期相对较长及其在化学发光混合物中的扩散，显示出信号定位问题。该系统已被证明具有高灵敏度，能够进行定量分析，并且相对简单。
