Peptide-based Methods For Detection Of Two Tumor Marker Proteins

Tumor markers play a significant role in early diagnosis, recurrence or metastasis detection, efficacy and prognosis evaluation in patients with cancer. Quantitative analysis of tumor markers is essential to the clinical management of cancer patients. Traditionally used methods for the detection of protein-markers are ELISA and immunohistochemistry, which rely on antibodies to recognize target protein. Although they have high specificity and sensitivity, some limitations remain. First, antibodies cannot easily be modified and synthesized due to its complex structure. Second, antibodies are commonly high cost and unstable. In recent years, the development of phage display technology has a profound impact on the research of mutual recognition between protein molecules. By virtue of phage display peptide library, many high affinity protein binding peptides are screened. The molecular structure of these peptides is simple and clear, and they can be artificially synthesized and modified according to different purposes. This characteristic is to some extent supplement to the limitations of antibodies. Herein, two kinds of methods for detection of tumor marker proteins based on the interaction between peptides and target proteins have been designed, which is meaningful to clinical management of cancer patients. 1. Peptide-based sensitive detection of melanoma biomarker S100B with electrochemical technique.Melanoma is one of the most aggressive and life-threatening forms of cancer, which is characterized by high risk of recurrence and mortality. Biomarker S100B is not only closely associated with melanoma burden and progression, but also can provide crucial clinical information about the prognostic and treatment response. So, we propose an electrochemical assay to detect S100B. This assay takes advantage of the 1:2 binding between S100B and its specific binding peptide. The first peptide immobilized on a gold electrode can capture S100B, while a second peptide modified with Gly-His-Lys copper ion chelating motif can generate signal. The chelated copper ion can also amplify the signal by catalyzing the oxidation of o-phenylenediamine (OPD) to electroactive3-diaminophenazine (DAP). By using square wave voltammetry, a limit of detection as low as 0.1 nM can be achieved. This method has also demonstrated favorable recovery in the complex serum sample, indicating potential clinical application in the future.2. Peptide-based colorimetric assay of bevacizumab response marker VEGFR-1.Targeted anti-angiogenesis is an important part of anti-cancer therapy, whose representative drug is bevacizumab. This drug has been approved by FDA for advanced colon cancer, kidney cancer, non-small cell lung cancer, HER-2 negative breast cancer and glioma. It has been reported that VEGFR-1 is a well predictor for treatment response of bevacizumab. In this work, we propose a novel peptide-based colorimetric method to analysis VEGFR-1. Peptides with N-terminal aromatic residue are first immobilized on gold nanoparticles (GNPs). Since cucurbit[8]urils (CB[8]) can selectively accommodate two N-terminal aromatic residue of peptides simultaneously, peptides on the GNPs can be dimerized by CB[8] and thus aggregation of GNPs can be formed. When peptide-coated GNPs are incubated with VEGFR-1 first, N-terminal aromatic residue of peptides are occupied due to the specific binding between VEGFR-1 and peptide, thus CB[8] cannot work and no aggregation can be formed. However, in the absence of VEGFR-1, peptides with N-terminal aromatic residue will be dimerized by CB[8] and further leads to the aggregation of peptide-coated GNPs. By virtue of UV-vis spectroscopy, protein-peptide binding event can be transduced into colorimetric signal. The detection limit of our method is down to 0.2 nM. The feasibility of our method has also demonstrated in spiked serum sample, indicating potential application in the future.