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A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe
摘要： The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5′ hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL?1), a low detection limit (0.01 U·mL?1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.
关键词： DNA-AgNCs,Cell extracts,Clinical diagnostics,Lambda exonuclease,Proximity effect,T4 PNK,Inhibitor,ATP,Na2HPO4,Phosphorylation
更新于2025-09-23 15:23:52
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An Enhanced Silver Nanocluster System for Cytochrome c Detection and Natural Drug Screening targeted for Cytochrome c
摘要： A label-free, ultra-sensitive and turn-off fluorescence method for detecting cytochrome c (Cyt c) has been recently developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs@tween 80) as a novel fluorescence-enhancing nanoprobe. Cyt c-induced electron transfer between Cyt c and DNA-AgNCs results in a significant fluorescence quenching effect of DNA-AgNCs. The coated tween 80 around DNA-AgNCs particles significantly caused fluorescence signal enhancement in the solution. Meanwhile, the efficiency of fluorescence quenching by Cyt c increases substantially by adding 0.01% tween 80 into the detection system. Based on these findings, a simple method with the detection limit of 0.8 nM was established for Cyt c based on tween 80 enhanced DNA-AgNCs. A good linear relationship between the fluorescent intensity and the concentration of Cyt c ranging from 0.8 nM to 10000 nM (R2=0.9910) was obtained. Consequently, the method was applied to monitor the Cyt c variation in HCT-116 and BGC-823 tumor cell culture medium after being treated with (doxorubicin) DOX and two kinds of natural compounds (J5 and S6, extracted from plants Abacopteris Penangiana and Swertia punicea Hemsl, respectively). The results indicated that the level of Cyt c in cell culture medium decreased according to the presence of DOX, J5 and S6. The reliability of the result was further confirmed by using cell viability assay and FACS analysis. From these data, it can be concluded that this groundbreaking nanoprobe with high sensitivity, great simplicity and good biocompatibility boasts a promising outlook for exploring the molecular mechanisms of apoptosis regulation and nature drug screening.
关键词： DNA-AgNCs,natural compounds,drugs screening,cytochrome c
更新于2025-09-23 15:22:29
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Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification
摘要： Abnormal concentration of adenosine triphosphate (ATP) is directly asscociate with several diseases. Thus, sensitive detection of ATP is essential to early diagnosis of disease. Herein, we described an ultrasensitive strategy for ATP detection by using positively charged gold nanorods ((+)AuNPs) as an efficient fluorescence quenching platform, coupled with exonuclease (cid:1) (Exo (cid:1)) assisted target recycling amplification. To construct the sensor, DNA template that contained ATP aptamer was used for the formation of AgNCs signal probe (DNA/AgNCs), the structure of it could change to duplex after the interaction of it with ATP. Such DNA template or duplex DNA product could electrostatically adsorb onto (+)AuNRs surface, resulting in the quenching of the fluorescence signal due to the vicinity of AgNCs to (+)AuNRs. With the addition of Exo (cid:1), DNA duplex could be hydrolyzed and released from (+)AuNRs surface, leading to the recovery of a strong fluorescent signal, while ATP could be regenerated for next target recycling. Combing the good fluorescence quenching ability of (+)AuNRs and the Exo (cid:1) assisted signal amplification, a low detection limit of 26 pM was achieved for ATP detection. Notably, the proposed method can be successfully applied for detecting ATP in serum samples, indicating a potential application value in early cancer diagnosis.
关键词： Exo (cid:1),Fluorescent sensor,(+)AuNRs,DNA/AgNCs,ATP detection,Target recycling amplification
更新于2025-09-16 10:30:52