AbstractThe protein mucin1 (MUC1) is an attractive target for cancer biomarkers because it is overexpressed in most adenocarcinomas. In this study, we exploited a MUC1-binding aptamer (Apt_MUC1) as a targeting agent for nanoparticle-based imaging systems coupled with laser desorption/ionization mass spectrometry (LDI-MS). We found that Apt_MUC1-conjugated gold nanoparticles immobilized, through hydrophobic and π–π interactions, on graphene oxide (Apt_MUC1–Au NPs/GO) bound effectively to MUC1 units on tumor cell membranes. The ultrahigh density and high flexibility of Apt_MUC1 on the GO surface enhanced the platform’s cooperative and multivalent binding affinity for MUC1 on cell membranes. After we had labeled MUC1-overexpressing MCF-7 cells (human breast adenocarcinoma cell line) with Apt_MUC1–Au NPs/GO, we used LDI-MS to monitor Au cluster ions ([Au_n]⁺; n = 1–3), resulting in the detection of as few as 100 MCF-7 cells. We also employed this Apt_MUC1–Au NPs/GO–LDI-MS system to analyze four different MUC1 expression cell lines. In addition, the Apt_MUC1–Au NPs/GO platform could be used further as a labeling agent for tumor tissue imaging when coupled with LDI-MS. Thus, Apt–Au NPs/GO can function as a highly amplified signal transducer through the formation of large Au clusters ions during LDI-MS analysis.