Development of a highly sensitive photometric readout platform technology using SPAAC-modified HRP liposomes

Liposome-based bioassays take advantage of liposomes' immense signaling power and easy surface modification. Photometric, electrochemical, and fluorescent detection methods dominate their application in bioassays. Here, it is studied how covalent modification of the liposome surface with proteins affects the functionality of entrapped enzymes. Horseradish peroxidase (HRP) was selected as a model for entrapment while streptavidin served as a model surface ligand. Several coupling reagents negatively affected the functionality of entrapped HRP. In the case of EDC-sNHS chemistry, membrane-permeable reagents inactivated HRP. Similarly, when applying copper-free click chemistry, in particular strain-promoted azide-alkyne cycloaddition (SPAAC), azide-functionalized lipids caused enzyme inactivation probably through coordination with the prosthetic heme iron of HRP. In contrast, the inverse approach employing DBCO-functionalized lipids fully preserved HRP activity. Under optimized conditions with coupling times of only 24 h at room temperature, streptavidin coupling efficiencies reached standard levels. Entrapped HRP was stabilized with 310 mM trehalose and 2 mg/mL HSA enabling storage for up to 6 months after streptavidin modification. HRP-loaded liposomes were evaluated by adapting an assay for the detection of SARS-CoV-2 neutralizing antibodies in serum. Therefore, the liposome surface was modified with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The RBD-modified liposomes with simple photometric detection demonstrated a similar sensitivity as fluorescent sulforhodamine B (SRB) liposomes, with a strong correlation of both methods (Pearson's r = 0.987). HRP liposomes are hence a reliable platform for visual or photometric bioassays providing similar limits of detection as fluorescent approaches making them ideal for routine lab and point-of-care applications.