研究目的
To develop and compare two fluorescent aptasensor methods (turn-on and ratiometric) for the sensitive detection of ATP in biochemical systems.
研究成果
The developed aptasensor methods are simple, sensitive, and selective for ATP detection, with low detection limits and good linear ranges. They offer advantages such as rapid analysis (within 1 hour), cost-effectiveness, and potential for adaptation to other small molecules. The ratiometric method provides self-calibration benefits for precise measurements in complex environments.
研究不足
The methods have detection limits in the micromolar range (3.5-3.8 μM), which may not be sufficient for very low ATP concentrations. The complexity of the ratiometric method involves labeling and conjugation steps, which could be time-consuming. Applicability in highly complex biological matrices beyond diluted human serum was not extensively tested.
1：Experimental Design and Method Selection:
The study designed two aptasensor methods: a label-free turn-on fluorescence approach and a FRET-based ratiometric approach. Both methods utilize the ATP-binding aptamer and berberine to induce G-quadruplex formation upon ATP binding, leading to fluorescence changes.
2：Sample Selection and Data Sources:
ATP aptamer sequences were obtained from Integrated DNA Technologies. ATP and its analogues (CTP, GTP, UTP, ADP, AMP) were purchased from Sigma-Aldrich. Human serum samples were used for spiking experiments.
3：List of Experimental Equipment and Materials:
Equipment includes a CARY 300 Bio spectrophotometer (Varian, USA) for UV-Vis, a fluorescence spectrophotometer (Horiba Jobin Yvon, USA), a Jasco J-815 spectropolarimeter for CD, FEI Tecnai F20 FETEM for TEM, and a high-speed centrifuge (Thermo Electron Corporation). Materials include berberine, gold nanorods (AuNRs), silver nanoparticles (AgNPs), red quantum dots (RQDs), carbon dots (CDs), and various chemicals from Sigma-Aldrich.
4：Experimental Procedures and Operational Workflow:
For the turn-on method, aptamer and berberine were mixed with ATP, incubated, and fluorescence measured. For the ratiometric method, aptamers were labeled with quenchers and fluorophores, mixed with ATP and berberine, incubated, and dual emissions monitored. CD, UV-Vis melting, and TEM analyses were performed to confirm structural changes.
5：Data Analysis Methods:
Fluorescence enhancement and FRET ratios were calculated. Detection limits were determined using linear regression (3.3 × Syx/slope). Statistical analysis included triplicate measurements.
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