In this work, a method for quantifying the activity of formamidopyrimidine DNA glucosylase (Fpg) was designed based on phosphate group (P)-modulated multi-enzyme catalysis and fluorescent copper nanoclusters (CuNCs). By eliminating 8-oxoguanine from double-stranded DNA, Fpg generates a nick with P at both 3′ and 5′ termini. Subsequently, part of the DNA is digested by 5′P-activated lambda exonuclease (λ Exo), and the generated 3′P disables exonuclease I (Exo I), resulting in the generation of single-stranded DNA containing poly(thymine) (poly(T)). Using poly(T) as templates, CuNCs were prepared to emit intense fluorescence as the readout of this method. However, in the absence of Fpg, the originally modified 5′P triggers the digestion of λ Exo. In this case, fluorescence emission is not obtained because CuNCs cannot be formed without DNA templates. Therefore, the catalysis of λ Exo and Exo I can be tuned by 5′P and 3′P, which can be further used to determine the activity of Fpg. The fluorescent Fpg biosensor works in a “signal-on” manner with the feature of “zero” background noise, and thus shows desirable analytical features and good performance. Besides, Fpg in serum samples and cell lysate could be accurately detected with the biosensor, indicating the great value of the proposed system in practical and clinical analysis.

中文翻译：

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基于磷酸基团调制的多酶催化和荧光铜纳米簇灵敏测定甲酰嘧啶DNA葡萄糖基化酶。

在这项工作中，基于磷酸盐基团（P）调节的多酶催化和荧光铜纳米簇（CuNCs），设计了一种定量甲酰嘧啶DNA葡萄糖基化酶（Fpg）活性的方法。通过从双链DNA中消除8-氧代鸟嘌呤，Fpg会在3'和5'末端产生一个带有P的切口。随后，部分DNA被5'P激活的λ核酸外切酶（λExo）消化，并且生成的3'P使核酸外切酶I（Exo I）失效，从而导致了含有多聚胸腺嘧啶的单链DNA（ poly（T））。使用poly（T）作为模板，制备了CuNC，以发出强烈的荧光，作为该方法的读数。但是，在没有Fpg的情况下，最初修饰的5'P触发了λExo的消化。在这种情况下，由于没有DNA模板就无法形成CuNC，因此无法获得荧光发射。因此，λExo和Exo I的催化作用可通过5'P和3'P进行调节，这可进一步用于确定Fpg的活性。荧光Fpg生物传感器以“信号开启”方式工作，具有“零”背景噪声的特征，因此显示出理想的分析特征和良好的性能。此外，利用生物传感器可以准确检测血清样品和细胞裂解物中的Fpg，这表明该系统在实际和临床分析中具有重要价值。