Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies

Reagentless immunosensors are emergingto addressthe challenge of practical and sensitive detection of important biomarkersin real biological samples without the need for multistep assays anduser intervention, with applications ranging from research tools topoint-of-care diagnostics. Selective target binding to an affinityreagent is detected and reported in one step without the need forwashing or additional reporters. In this study, we used a structure-guidedapproach to identify a mutation site in an antibody fragment for thepolarity-dependent fluorophore, Anap, such that upon binding of theprotein target cardiac troponin I, the Anap-labeled antibody wouldproduce a detectable and dose-dependent shift in emission wavelength.We observed a significant emission wavelength shift of the Anap-labeledanti-cTnI mutant, with a blue shift of up to 37 nm, upon binding tothe cTnI protein. Key differences in the resulting emission spectrabetween target peptides in comparison to whole proteins were alsofound; however, the affinity and binding characteristics remainedunaffected when compared to the wild-type antibody. We also highlightedthe potential flexibility of the approach by incorporating a near-infrareddye, IRDye800CW, into the same mutation site, which also resultedin a dose-dependent wavelength shift upon target incubation. Thesereagents can be used in experiments and devices to create simplerand more efficient biosensors across a range of research, medicallaboratory, and point-of-care platforms.

Automated summary

Reagentless immunosensors are being developed for practical and sensitive detection of biomarkers in biological samples without the need for multistep assays. This study used a structure-guided approach to identify a mutation site in an antibody fragment for polarity-dependent fluorophores, allowing for detectable wavelength shifts upon binding to the protein target. The results showed significant emission wavelength shifts and demonstrated the potential of incorporating different dyes into the same mutation site. These reagents have applications in research, medical laboratories, and point-of-care platforms to create simpler and more efficient biosensors.