Immunofluorescence is a widely used technique to detect proteins inside cells or tissues in vitro using antibodies. Due to their size and net charge, antibodies are unable to enter the cells by passive transport, thus cell fixation by chemical compounds (like aldehydes, methanol, etc.) and subsequent permeabilization of the plasmatic membrane become necessary to target intracellular proteins (Im et al., 2019). Indirect fluorescence staining requires a two-steps delivery of antibodies: first, a primary antibody is delivered into the cells where it recognizes the protein of interest; then a fluorophore-labeled secondary antibody allows for the visualization of the protein, thanks to its specific link to the primary antibody. Nowadays, many primary antibodies conjugated with fluorescent probes or other fluorescent markers are commercially available, which may reduce the immunostaining protocol to one step only (direct fluorescence; Bishop et al., 2018; Im et al., 2019).

In the past two decades, new techniques emerged, which partially or totally avoid fixation and thus realize the so-called live immunofluorescence or live imaging of both primary cells and cell lines in vitro (Kang et al., 2016). Among these techniques, single cell microinjection was successfully used to deliver a polyclonal anti-actin antibody inside rat embryonic astrocytes in culture, to study the influence of actin on gap junctions-mediated intercellular communication. In this case, however, fixation was needed after the microinjection of the primary antibody, in order to deliver the fluorescent secondary antibody (Theiss and Meller, 2002). Similarly, fixation was necessary to vehicle the secondary antibody in murine and human cell lines after a primary anti-tubulin antibody was introduced in live cells by means of an envelope vector system based on hemagglutinating virus of Japan (Kondo et al., 2008). Other groups microinjected rhodamine-conjugated actin and Phalloidin in live fibroblasts (Wang, 1987) and smooth muscle BC3H1 cells (Sund and Axelrod, 2000) in vitro without fixation, thus studying dynamics of both G- and F-actin. In live HeLa cells, a fluorescent primary antibody directed against the Golgi protein giantin was successfully delivered by a cationic lipids-based agent, resulting in a specific and tight interaction with the antigen, which resisted to the subsequent permeabilization of the cells with digitonine (Weill et al., 2008). Similarly, polimersomes were used to vehicle FITC-labeled monoclonal anti-α-tubulin antibody into live primary human dermal fibroblasts and CHO cell line (Massignani et al., 2010). Other researchers took advantage of plasmonic nanoparticles to deliver monoclonal anti-actin antibodies into live NIH3T3 fibroblasts and study intracellular actin dynamics during cellular motility (Kumar et al., 2007). The pore-forming property of the bacterial toxin Streptolysin O, instead, was exploited to deliver fluorescent Phalloidin and antibodies into CHO-K1 and HeLa cells (Teng et al., 2016). Recently, fluorescent labeled nanobodies, which are the smallest antigen-binding fragments, were used to target the cytoskeletal proteins fascin and vimentin in living HeLa cells by means of a laser beam-based photoporation (Liu et al., 2018; Liu et al., 2020). The same technique was used to deliver anti-cortactin and anti-β catenin nanobodies for live imaging in HeLa and HNSCC61 cells (Hebbrecht et al., 2020). Also, cell-permeant bioadaptors were successfully used to deliver intracellular antibodies in HeLa cells and study the oxidative stress through live cells imaging of protein glutathionylation (Du et al., 2020). In primary hippocampal neurons of rodents, which are notably challenging to transfect, fluorescent IgG were efficiently delivered through a synthetic protein transduction domain mimic, requiring a four hour incubation on live cells (Backlund et al., 2020).

Besides the aforementioned, electroporation is one of the most used and efficient techniques to deliver antibodies into living cells in vitro since many years ago (Campbell et al., 1995). Standard electroporation in suspension allowed to deliver primary antibodies conjugated with fluorescent dyes into mammalian cells (Berglund & Starkey, 1991; Conic et al., 2018; Conic et al., 2019), also targeting nuclear compartments (Freund et al., 2013), and to realize single-molecule speckle microscopy detection of actin (Yamashiro and Watanabe, 2017). Electroporation was also applied to improve the delivery of monoclonal antibodies conjugated with TiO2 nanoparticles into human LoVo cancer cells, where they were exploited as therapeutic agents (Xu et al., 2007).

In parallel to bulk electroporation, in situ electroporation of adherent cells is extensively used as a mean to target intracellular antigens. A single pulse of 150 V and 20 ms duration was applied to HeLa cells to deliver a fluorescent monoclonal antibody involved in the apoptotic pathway (Rui et al., 2002). Recently, a graphene-based substrate was developed to electroporate and image live adherent epithelial cell lines, primary hippocampal neurons and stem cells with organic dyes like fluorescent Phalloidin and fluorophore-conjugated anti-vimentin and -spectrin primary antibodies, reaching high electroporation efficiencies by using low voltage range (15 V) and halftime (5–10 ms) pulses (Moon et al., 2020). To characterize adhesion and cell migration in NIH/3 T3 fibroblasts in culture, the combined delivery of Phalloidin and a vinculin-coding plasmid has been realized through subsequent electroporations on glass-integrated aluminum electrodes (Zhang et al., 2020).

In this context, we used thin film SiO2 capacitive microelectrodes, recently shown to be reliable devices for DNA transfection of mammalian cell lines and primary neuronal cells (Maschietto et al., 2021), to deliver fluorescent-conjugated organic dyes and antibodies into live cell cultures through electroporation. Stimuli with a very low voltage range (±3 V) compared to other devices allowed to reach high electroporation efficiencies (up to about 80–90% for antibodies), avoiding chemical artifacts and time-consuming procedures required by fixation, but at the same time preserving antibody structure and specificity and cells integrity. We demonstrated this application by using a fluorescence-conjugated antibody and the toxin Phalloidin, both directed against actin which is one of the most important proteins involved in adhesion and motility of mammalian cells. The work opens the possibility to employ these microchips to study any intracellular protein of interest.