Competitive aptasensor for the ultrasensitive multiplexed detection of cancer biomarkers by fluorescent nanoparticle counting

Cancer biomarker quantification in human serum is of great importance for accurate patient diagnosis and informed clinical management. To date, ultrasensitive multiplexed detection of proteins without amplification is still a major challenge. Herein, we proposed a competitive aptasensor strategy for ultrasensitive multiplexed cancer biomarker detection by fluorescent nanoparticle (FNP) counting. The sequences are designed such that the binding abilities of linker DNA (L-DNA) with DNA-functionalized FNPs (DNA-FNPs) and aptamer are comparable. As long as one target binds with one molecule of aptamer, a signalling FNP forms a sandwich-structured nanocomposite, which was subsequently observed and enumerated with a fluorescence microscope. This 1 : 1 target-to-signal FNP production assured an improved sensitivity, benefiting from the reasonably good brightness and photostability of FNPs. For both singleplexed and multiplexed detection, this proposed strategy achieved an approximately 1000-fold improved limit of detection than the conventional method with the detection volume of 3.2 μL. Notably, the results for carcinoembryonic antigen (CEA) detection obtained directly from 9 human serum samples (colorectal/lung/healthy individuals) were consistent with that obtained by ELISA, showing potential application in clinical diagnosis.