Nanocounter on Cell Membrane: In-Situ Quantification of Membrane-Bound Enzymes with High Simplicity and Sensitivity via the Assembly of Branched Peptides

The quantitative analysis of membrane-bound enzymes (MBEs) is still challenged by complicated process, expensive equipment, and/or limited sensitivity. Herein, a highly sensitive yet user-friendly colorimetric method is proposed for the in-situ quantification of MBEs via the reasonable assembly of branched peptides. Taking alkaline phosphatase (ALP) as an example, the designed assembly of branched peptides can respond to the membrane-bound enzyme and undergo a spontaneous transformation to load signal reporters, giving rise to a colorimetric signal. Therefore, this method allows for simple in-situ measurements of MBEs without the need for complicated protein isolation or labeling processes or sophisticated equipment. Significantly, the exploitation of branched peptides with multiple functional sites enables both multivalent recognition of targets and large-scale loading of signal reporters, achieving ultra-high sensitivity. As a result, as few as 34pg of ALP on HepG2 cell membrane can be measured directly in cell suspension in an add-to-respond manner. Furthermore, the successful differentiation of cancer cells with ALP overexpression depicts the potential application of this method. By adjusting the target-responsive peptide sequences, this work can be generally expanded for versatile MBE analysis and other related biomedical research.