1：Experimental Design and Method Selection:

The method is based on a turn-off fluorometric approach using DNA-templated silver nanoclusters (AgNCs). It involves the phosphorylation of DNA probes by T4 PNK, followed by cleavage with Lambda exonuclease (λ exo), leading to separation of G-rich and C-rich sequences and a decrease in fluorescence.

2：Sample Selection and Data Sources:

Oligonucleotide sequences (DNA S1 and DNA S2) were synthesized and used. T4 PNK, λ exo, ATP, and other reagents were purchased from commercial sources. Cell extracts from PC-3 M cells were used for practical applications.

3：List of Experimental Equipment and Materials:

Equipment includes a PerkinElmer LS-55 fluorescence spectrometer and field emission transmission electron microscopy. Materials include DNA sequences, T4 PNK, λ exo, ATP, silver nitrate, sodium borohydride, Tris-HCl buffer, MgCl2, DTT, fetal calf serum, DMEM medium, and Na2HPO

4：Experimental Procedures and Operational Workflow:

DNA-AgNCs were synthesized and hybridized with DNA S2. The phosphorylation reaction was performed in Tris-HCl buffer with MgCl2, DTT, ATP, λ exo, and T4 PNK at 37°C for 30 min, followed by heat inactivation. Fluorescence was measured at 650 nm with excitation at 580 nm. Cell assays involved culturing cells, lysing them, and applying the same detection procedure.

5：The phosphorylation reaction was performed in Tris-HCl buffer with MgCl2, DTT, ATP, λ exo, and T4 PNK at 37°C for 30 min, followed by heat inactivation. Fluorescence was measured at 650 nm with excitation at 580 nm. Cell assays involved culturing cells, lysing them, and applying the same detection procedure. Data Analysis Methods:

5. Data Analysis Methods: Fluorescence intensities were measured and analyzed to determine T4 PNK activity, with linear range and detection limit calculated based on signal-to-noise ratio. Inhibition studies used Na2HPO4, and recoveries in cell extracts were evaluated.