Functional mass nanoprobes inserted on live cells for in situ monitoring multiple secreted enzymes with MALDI-TOF mass spectrometry

Numerous enzymes secreted from tumor cells have been used as biomarkers for early diagnosis, but in situ monitoring their secretion is still a challenge owing to the low amount and diffusion into complex environment. Herein, we establish a mass spectrometric protocol for in situ dynamic biosensing of multiplexed enzyme secretions by inserting tentacle-like mass nanoprobes on live cells. The nanoprobes are designed to contain an inserting group for cell anchoring and abundant substrate peptides for mass coding to facilely capture the secreted target enzymes followed with in situ MALDI-TOF MS detection. From the peaks of the substrates and their cleavage products, the quantification strategy of the corresponding secreted enzyme is achieved. Taking MMP-2 and MMP-9 as targets, the in situ monitoring protocol is demonstrated to provide a high-throughput technique for rapid detection of multiple secreted enzymes. Its universality for more secreted enzymes is also verified with cell-secreted urokinase-type plasminogen activator and alkaline phosphatase. The results identical to those from conventional phenotype methods for assessment of drugs efficacy indicate the promising application of the proposed protocol.