研究目的
To develop an ultrasensitive electrochemiluminescence biosensor for detecting protein kinase A activity and screening inhibitors, leveraging Au nanoparticles for signal amplification.
研究成果
The ECL biosensor based on Au NPs amplification provides highly sensitive detection of PKA activity with a low LOD of 0.0002 U/mL, outperforming previous methods. It demonstrates good stability, reproducibility, and selectivity, and is effective for inhibitor screening and real sample analysis, showing promise for clinical diagnostics.
研究不足
The method may be specific to PKA and might not generalize to other kinases without modification. The use of Au nanoparticles could introduce variability in synthesis and immobilization. Real sample analysis was limited to spiked serum, and potential interferences in complex biological matrices were not fully explored.
1：Experimental Design and Method Selection:
The biosensor was designed using Au nanoparticles to enhance the ECL signal of the S2O8 2?–O2 system. The method involves immobilizing peptides on a chitosan-modified glassy carbon electrode, phosphorylating them with PKA and ATP-s, capturing Au nanoparticles via Au-S bonds, and measuring ECL intensity.
2：Sample Selection and Data Sources:
Peptide H2N-LRRASLGGGGR-OH was used. PKA and ATP-s were employed for phosphorylation. Human serum samples from the First Affiliated Hospital of Nanchang University were used for real sample analysis.
3：List of Experimental Equipment and Materials:
Glassy carbon electrode (GCE), chitosan (CS), N-hydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), peptide, ATP-s, PKA, Au nanoparticles (average diameter 15 nm), Na2S2O8, Tris-HCl buffer, MgCl2, PBS buffer.
4：Experimental Procedures and Operational Workflow:
Clean GCE, coat with CS, dip into EDC/NHS and peptide solution for 10 h, immerse in Tris-HCl buffer with MgCl2, PKA, and ATP-s for 100 min, wash, dip into Au NPs solution for 1 h, wash, characterize with ECL in PBS with Na2S2O8 at -1.8 to 0 V potential range.
5：8 to 0 V potential range.
Data Analysis Methods:
5. Data Analysis Methods: ECL intensity was measured and correlated with PKA concentration. Statistical analysis included linear regression for calibration curve, calculation of limit of detection (LOD), and relative standard deviation (RSD) for reproducibility.
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