Turning Nonspecific Interference into Signal Amplification: Covalent Biosensing Nanoassembly Enabled by...

In this work, a new method of protein detection in complicated samples is proposed. This method employs probe-target recognition to induce cross-linking among the probe, the target, and the nonspecific proteins in the complicated sample as a means to convert interference into effective signal amplification. This also eliminates the necessity of multistep signal amplification in a separate solution system. On the basis of this strategy, a simple and robust assay for the activity of serum cathepsin B is established. Peptide probes immobilized on a sensing slide can recognize cathepsin B, and this can induce thiol-alkyne covalent coupling between the probe and cathepsin B. Meanwhile, applying electrochemical potential scanning to this sensing surface, Cu binding fragments of the probe peptide can be released into the solution phase to act as an electrochemical catalyst for oxidative dityrosine cross-linking among all proteins including the captured cathepsin B and the nonspecific proteins. A continuous nanoassembly covalently anchored on the sensing surface can gradually form, allowing violent detergent rinsing to remove residual interference. Using this method, not only sensitivity in the picomolar range can be achieved for serum analysis, the results of the analysis can also reliably discriminate benign and cancerous ovarian conditions. These results may suggest prospective application of this method in early screening of cancer in the future.