## 4 Conclusions  
In this work, we devised a method based on miniaturized F4-multidetection to size-separate blood serum into four components (SA and size equivalents, IgG and 200-300 kDa immunoglobulins, IgM, lipoprotein particles), in order to study the interactions between heme and each subpopulation in a native and representative state. The experiments were carried out in PBS and required a short analysis time (<30 min) and very little sample injection volumes. As low as 0.5 uL of serum could be injected, and the presence of bound heme could be selectively monitored thanks to the removal of free heme during the method's focusing step. The main interacting protein was found to be serum albumin, while a low amount of heme also bound to other proteins such as IgM. The ability/inability to bind heme in physiological conditions was also verified individually for SA and IgG, the latter being uncapable to bind heme at clinically relevant concentrations. To simulate co-morbidity and explore different concentration equilibria and competitions, we also employed an IgG-enriched serum, yielding the same results. This approach proved to be extremely information-rich, since it allows for mass and size characterization, subcomponent separation and monitoring of deviations of the concentration of serum's most abundant proteins. Since the formation of stable complexes between blood proteins and heme is not fully understood, this setup can contribute to increase the knowledge on preferred heme-clearing pathways. Moreover, this approach serves also as a proof-of-concept for further studies selectively monitoring other small molecules and probes of clinical/pharmaceutical interest. This is particularly relevant for complex samples such as serum, since it preserves native conditions and avoids recurring to time and lab-heavy processing steps. Lastly, this approach could also be translated towards the study of selective heme-antibody binding to investigate specific interactions at the native state, preserving spatial arrangements.  
The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app12136762/s1](https://www.mdpi.com/article/10.3390/app12136762/s1)]([https://www.mdpi.com/article/10.3390/app12136762/s1](https://www.mdpi.com/article/10.3390/app12136762/s1)), Figure S1: platform schematics and method output; Figure S2: Negative control of heme alone in in HF5-multidetection.  
Conceptualization, V.M., A.Z., S.G. and B.R.; Data curation, S.G.; Funding acquisition, A.Z. and P.R.; Investigation, V.M. and S.G; Methodology, V.M., S.G., A.Z., P.R. and B.R.; Supervision, A.Z. and V.M.; Writing--original draft, V.M., A.Z. and S.G.; Writing--review and editing, V.M., S.G., A.Z., P.R. and B.R. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
Not applicable.  
V.M., B.R., P.R. and A.Z. are associates of the academic spinoff company byFlow SRL (Bologna, Italy). The company mission includes know-how transfer, development, and application of novel technologies and methodologies for the analysis and characterization of samples of nano-biotechnological interest.