Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5′ end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5′-end and biotin modification at 3′-end). The formed blunt 5′-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.

基于链霉亲和素包被的纳米球和T7核酸外切酶辅助的双周期信号放大，我们开发了一种新颖的miRNA敏感荧光偏振检测方法。当目标miRNA存在于系统中时，发夹探针与miRNA杂交，形成双链结构。然后识别发夹探针的5'端并通过T7核酸外切酶消化，释放未降解的单链DNA片段和miRNA。释放的靶标miRNA可能触发杂交和消化的下一个周期，从发夹探针释放更多未降解的片段。这些片段可以与信号探针杂交（在5'末端修饰羧基荧光素，在3'末端修饰生物素）。然后通过T7核酸外切酶识别并降解形成的信号探针平端5'-末端，释放片段和荧光团羧基荧光素。信号探针杂交和消化的下一个周期同时被释放的片段触发。游离的羧基荧光素不能与用作荧光偏振信号放大器的抗生蛋白链菌素包被的纳米球连接。因此，由于捕获的纳米球完整信号探针与游离羧基荧光素之间的荧光偏振信号不同，在将miRNA加入检测系统后，荧光偏振信号发生了很大变化。该方法的检出限约为0.001 nM，具有良好的选择性。此外，它还可用于确定总miRNA提取物中的目标miRNA，并比较目标miRNA在不同细胞中的表达水平。所以，