Sensitive determination of formamidopyrimidine DNA glucosylase based on phosphate group-modulated multi-enzyme catalysis and fluorescent copper nanoclusters
Li, JY (Li, Junyao)[ 1,2 ] ; Zhang, MY (Zhang, Mengyang)[ 1,2 ] ; Wang, HS (Wang, Huaisheng)[ 3 ] ; Wu, J (Wu, Jie)[ 1,2 ] ; Zheng, RX (Zheng, Ruixue)[ 1,2 ] ; Zhang, JH (Zhang, Jiahui)[ 1,2 ] ; Li, Y (Li, Yan)[ 1,2 ] ; Wang, ZY (Wang, Zhaoyin)[ 1,2 ]*（王兆寅）; Dai, ZH (Dai, Zhihui)[ 1,2 ]*（戴志晖）
[ 1 ] Nanjing Normal Univ, Jiangsu Collaborat Innovat Ctr Biomed Funct Mat, Sch Chem & Mat Sci, Nanjing 210023, Peoples R China
[ 2 ] Nanjing Normal Univ, Jiangsu Key Lab Biofunct Mat, Sch Chem & Mat Sci, Nanjing 210023, Peoples R China
[ 3 ] Liaocheng Univ, Dept Chem, Liaocheng 252059, Shandong, Peoples R China
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 (lambda 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 lambda Exo. In this case, fluorescence emission is not obtained because CuNCs cannot be formed without DNA templates. Therefore, the catalysis of lambda 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.