荧光成像经常用于检测重要的目标分析物和实时、非侵入性监测生物学过程。近红外荧光探针由于其对生物组织的光损害小、有很深的组织穿透能力、受背景自发荧光干扰小等特点，而有利于活体成像。与单淬灭的荧光探针相比，双重淬灭的纳米荧光探针可以进一步降低背景荧光信号，从而在目标分析物的部位进一步提高了荧光的信噪比。然而，普通纳米探针通常面临着摄取低和靶向难的问题，此外还有制备繁琐和重现性差等问题。本项目拟合成两组基于FITC和Cy5.5的单淬灭荧光小分子探针，该探针在活细胞内谷胱甘肽还原下“智能”组装双重淬灭荧光纳米探针。在癌细胞内弗林蛋白酶的特异性剪切作用下，纳米探针释放出自由的FITC和Cy5.5片断，解除双重淬灭的效应，得到高信噪比的荧光信号，用于体内外弗林蛋白酶活性的特异性、高灵敏检测。由于弗林酶的高度表达是某些癌症早期发生的信号，因此本项目的双重淬灭荧光纳米探针将用于某些癌症的早期诊断。

Fluorescence imaging is always applied for the detection of important analytes and noninvasive visualization of biological processes in real time. Fluorescent probes in the near infrared (NIR) region are favorable for in vivo bioimaging because of their minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence. Compared with single quenched fluorescent probes, dual quenched fluorescent nanoprobes can further reduce the background fluorescence and therefore boost the signal-to-noise ratios in the region of interest. Nevertheless, premade nanoprobes are facing the problems of cell membrane translocation and targeting, besides the difficulty and reproducibility of their fabrications. In this proposal, we aim to design two FITC- and Cy5.5-based single quenched fluorescent probes. After entering cells, these probes will subject to glutathione-reduction-controlled “smart” self-assembling into dual quenched nanoprobes. Under the specific cleavage of furin in cancer cells, the nanoprobes will release the free FITC or Cy5.5 and relieve the dual-quenched effect, resulting in high signal-to-noise (S/N) ratio of the fluorescence. This fluorescence signal with high S/N ratio will be applied for specific detection of furin activity in vitro and in vivo with high sensitivity. Since overexpression of furin associates with the early development of some cancers, these dual quenched fluorescent nanoprobes in this proposal will be further developed for the early diagnosis of cancers.