研究目的
To construct a split aptamer based fluorescence resonance energy transfer (FRET) platform for the determination of adenosine deaminase (ADA) activity.
研究成果
The split aptamer based FRET platform successfully detected ADA with high sensitivity (LOD 0.72 U L-1) and selectivity, demonstrating applicability in human serum samples. It offers a simple, label-free alternative to existing methods, with potential for clinical diagnostics.
研究不足
The method may have limitations in complex biological matrices beyond serum, and the specificity was tested against a limited set of enzymes; further validation with more interferents might be needed. Optimization of conditions (e.g., reaction times, concentrations) was specific to this setup and may not generalize without adjustment.
1：Experimental Design and Method Selection:
The study designed a FRET-based sensing platform using split ATP aptamers (P1 and P2) conjugated to gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) for ADA detection. ATP binding brings donor (P1-AuNCs) and acceptor (P2-AuNPs) close for FRET quenching, and ADA catalyzes ATP to ITP, releasing aptamers and recovering fluorescence.
2：Sample Selection and Data Sources:
Human serum samples were used for real sample analysis, spiked with known ADA concentrations.
3：List of Experimental Equipment and Materials:
Materials included ADA, ATP, enzymes (pepsin, urease, lysozyme, tyrosinase, thrombin, glucose oxidase), THPC, 11-MUA, NHS, EDC, HAuCl4·4H2O, sodium citrate, and synthesized DNA aptamers P1 and P
4：Equipment included a RF-5301 PC spectrofluorophotometer (Shimadzu), Hitachi H-800 electron microscope for TEM/HRTEM, and Varian GBC Cintra 10e UV-visible Spectrophotometer. Experimental Procedures and Operational Workflow:
AuNCs were synthesized using THPC and 11-MUA, then conjugated with P1 via EDC/NHS reaction. AuNPs were synthesized via citrate reduction, then conjugated with P2 via Au-S bond. For ADA detection, ADA and ATP were incubated, followed by addition of P1-AuNCs and P2-AuNPs, with fluorescence measured after 10 min. Serum samples were processed similarly.
5：Data Analysis Methods:
Fluorescence intensity changes were measured, with linear regression used for ADA quantification, and recovery studies and RSD calculations for validation.
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