Streamlined integrated protein isoelectric focusing using microfluidic paper-based device.

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Electrokinetic elucidation of the interactions between persistent luminescent nanoprobes and the binary apolipoprotein-E/albumin protein system.

The affinity between functional nanoparticles (NPs) and proteins could determine the efficacy of nanoprobes, nanosensors, nanocarriers, and many other devices for biomedical applications. Therefore, it is necessary to develop analytical strategies to accurately evaluate the magnitude of these protein corona interactions in physiological media. In this work, different electrokinetic strategies were implemented to accurately determine the interactions between PEGylated ZnGa1.995Cr0.005O4 persistent luminescent NPs (ZGO-PEG) and two important serum proteins: human serum albumin (HSA), the most abundant serum protein, and apolipoprotein-E (ApoE), associated with the active transport of NPs through the blood-brain barrier. Firstly, the injection of ZGO-PEG in a background electrolyte (BGE) containing individual proteins allowed an affinity study to separately characterize each NP-protein system. Then, the same procedure was applied in a buffer containing a mixture of the two proteins at different molar ratios. Finally, the NPs were pre-incubated with one protein and thereafter electrokinetically separated in a BGE containing the second protein. These analytical strategies revealed the mechanisms (comparative, cooperative or competitive systems) and the magnitude of their interactions, resulting in all cases in notably higher affinity and stability between ZGO-PEG and ApoE (Ka = 1.96 ± 0.25 × 1010 M-M) compared to HSA (Ka = 4.60 ± 0.41 × 106 M-M). For the first time, the inter-protein ApoE/HSA interactions with ZGO-PEG were also demonstrated, highlighting the formation of a ternary ZGO-PEG/ApoE/HSA nanocomplex. These results open the way for a deeper understanding of the protein corona formation, and the development of versatile optical imaging applications for ZGO-PEG and other systemically delivered nanoprobes ideally vectorized to the brain.