Immunofluorescence imaging of cells plays a vital role in biomedical research and clinical diagnosis. However, when it is applied to relative quantification of proteins, it suffers from insufficient fluorescence intensity or partial overexposure, resulting in inaccurate relative quantification. Herein, we report a computer-aided design of DNA self-limited assembly (CAD-SLA) technology and apply it for relative quantification of membrane proteins, a concept proposed for the first time. CAD-SLA can achieve exponential cascade signal amplification in one pot and terminate at any desired level. By conjugating CAD-SLA with immunofluorescence, in situ imaging of cell membrane proteins is achieved with a controllable amplification level. Besides, comprehensive fluorescence intensity information from fluorescent images can be obtained, accurately showing relative quantitative information. Slight protein expression differences previously indistinguishable by immunofluorescence or Western blotting can now be discriminated, making fluorescence imaging-based relative quantification a promising tool for membrane protein analysis. From the perspectives of both DNA self-assembly technology and immunofluorescence technology, this work has solved difficult problems and provided important reference for future development.

中文翻译：

---

用于膜蛋白相对定量的 DNA 自限组装的计算机辅助设计

细胞的免疫荧光成像在生物医学研究和临床诊断中起着至关重要的作用。然而，当它应用于蛋白质的相对定量时，会出现荧光强度不足或部分过度曝光的问题，从而导致相对定量不准确。在这里，我们报告了 DNA 自限组装 (CAD-SLA) 技术的计算机辅助设计，并将其应用于膜蛋白的相对定量，这是首次提出的概念。CAD-SLA 可以在一锅内实现指数级联信号放大，并在任何所需水平终止。通过将 CAD-SLA 与免疫荧光结合，以可控的放大水平实现细胞膜蛋白的原位成像。此外，还可以从荧光图像中获得全面的荧光强度信息，准确显示相对定量信息。现在可以区分以前通过免疫荧光或蛋白质印迹无法区分的轻微蛋白质表达差异，使基于荧光成像的相对定量成为膜蛋白分析的有前途的工具。该工作从DNA自组装技术和免疫荧光技术两个角度解决了难题，为今后的发展提供了重要参考。