Bioconjugation of IgG Secondary Antibodies to Polymer Dots for Multicolor Subcellular Imaging

Abstract

Multiplexed subcellular imaging has attracted widespread attention in biology and medicine. Because the fluorescence brightness and stability of organic dyes and fluorescent proteins are not sufficient, the development of fluorescent nanoparticles is of broad interest. Semiconducting polymer dots (Pdots) exhibit prominent luminescence properties. However, the Pdot-IgG bioconjugates are largely unexplored in biological imaging. Here, we developed multicolor Pdots covalently conjugated with IgG secondary antibodies for specific labeling of different subcellular structures. By comparing the two functional polymers, we found that the functionalization by poly(styrene-co-maleic anhydride) (PSMA) could reliably produce Pdot-IgG conjugates for specific subcellular labeling, while those by a comb-like amphiphilic polymer show strong nonspecific binding in an intracellular environment. In addition, we minimize nonspecific adsorption of the Pdot bioconjugates by using bovine serum albumin and poly(ethylene glycol) as passivation agents, respectively. We finally demonstrate that the Pdot-PSMA-IgGs hold great potential in multiplexed visualization of the subcellular structure.

Expansion Microscopy with Multifunctional Polymer Dots

“…To facilitate multicolor staining with Pdots, we recently developed Pdot‐IgG bioconjugates for targeting subcellular structures. [ 15 ] We further investigated the performance of MA‐Pdots‐IgG bioconjugates by applying them to the visualization of the synaptic structures in the hippocampal cells of C57BL/6 mice. Pre‐ and post‐synaptic scaffolding proteins, namely, Bassoon and Homer, were labeled with MA‐PFO‐anti‐Mouse IgG and MA‐PFBT‐anti‐Rabbit IgG, respectively.…”

Near‐Infrared‐Emitting Boron‐Difluoride‐Curcuminoid‐Based Polymers Exhibiting Thermally Activated Delayed Fluorescence as Biological Imaging Probes

“…We chose an indirect immunolabeling strategy with a secondary antibody (see Figure 7 A) instead of direct immunolabeling because we anticipated less steric hindrance from the Pdot toward binding to HER2 on the cell membrane. Other studies with Pdots have also utilized this strategy or a similar one [51–53] . After conjugation with secondary antibodies, the mean hydrodynamic diameters of the CzBN‐ co ‐DtaB and CzBN‐ co ‐HmatB Pdots increased by ≈26–28 nm (Figure S24).…”

Near‐Infrared‐Emitting Boron‐Difluoride‐Curcuminoid‐Based Polymers Exhibiting Thermally Activated Delayed Fluorescence as Biological Imaging Probes

“…We chose an indirect immunolabeling strategy with as econdary antibody (see Figure 7A)i nstead of direct immunolabeling because we anticipated less steric hindrance from the Pdot toward binding to HER2 on the cell membrane.O ther studies with Pdots have also utilized this strategy or as imilar one. [51][52][53] After conjugation with secondary antibodies,t he mean hydrodynamic diameters of the CzBN-co-DtaB and CzBNco-HmatB Pdots increased by % 26-28 nm (Figure S24). This size increase was also confirmed by agarose gel electrophoresis.I ts hould be noted that the Pdot size distribution widened with an increase in average hydrodynamic diameter…”