Lab-on-chip microscope platform for electro-manipulation of a dense microtubules network

Pulsed electric field (PEF) technology is promising for the manipulation of biomolecular components and has potential applications in biomedicine and bionanotechnology. Microtubules, nanoscopic tubular structures self-assembled from protein tubulin, serve as important components in basic cellular processes as well as in engineered biomolecular nanosystems. Recent studies in cell-based models have demonstrated that PEF affects the cytoskeleton, including microtubules. However, the direct effects of PEF on microtubules are not clear. In this work, we developed a lab-on-a-chip platform integrated with a total internal reflection fluorescence microscope system to elucidate the PEF effects on a microtubules network mimicking the cell-like density of microtubules. The designed platform enables the delivery of short (microsecond-scale), high-field-strength (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le$$\end{document}≤ 25 kV/cm) electric pulses far from the electrode/electrolyte interface. We showed that microsecond PEF is capable of overcoming the non-covalent microtubule bonding force to the substrate and translocating the microtubules. This microsecond PEF effect combined with macromolecular crowding led to aggregation of microtubules. Our results expand the toolbox of bioelectronics technologies and electromagnetic tools for the manipulation of biomolecular nanoscopic systems and contribute to the understanding of microsecond PEF effects on a microtubule cytoskeleton.


List of supplementary materials with legend
• S1: this document • S2 -S7: microscopy videos, see Table S1.1 lower for details • S8: .xls file with analytical calculation of Debye length and electric field effect on detachment  2. Image analysis and measure development details
All videos are from TIRF imaging as described in the main text. For the analysis in the main text, we used video files 1-7 (experimental day 20200909 -formatted as YYYYMMDD). The further videos are: video file 9 from experimental day 20200731 and video file 11 from experimental day 20200819.

Supplementary videos
Selected videos are also provided as supplementary videos in .mp4 format, see Table 1. Data from videos S6 and S7 were not used for any of the quantitative analysis in the manuscript, they are just to demonstrate the detachment of microtubules from the substrate under the effect of PEF, when there was no methyl cellulose in the sample. Figure S1.2: Illustration of the mechanism behind the measure microtubule displacement index: TIRF microscope intensities prior (A) and after (B) µs-PEF, a difference of intensity matrices (C) with appearance of MTs at the red and yellow spots and disappearance of MTs at the blue spots, and a difference of absolute values of intensity matrices (D) that was used in the formula in order to cover the both types of changes.

Debye length calculation
The Debye length, a measure of the distance at which the3 electric potential from a source will decrease to 1/e due to screening of mobile charges, is given as [1] where is the relative permittivity of the medium (here we consider water-like, i.e. =78), 0 is the permittivity of the vacuum, k is Boltzmann constant, T is temperature (considering 298 K), ρ i is the number density of the charge (ion), z i is the ion valency e is elementary charge. The Debye length can be also expressed as [2] λ D = 0 kT 2e 2 N A I (S1.2) where N A is Avogadro's constant, and I is ionic strength [3][p.259] Figure S1.3: Clarification of microtubule overlap rate: Histograms of TIRF intensities prior and after µs-PEF for 4 lower voltages.
where c i is concentration of the ion in mol/m 3 . Expression in Eq. S1.2 is useful because I can be approximately obtained from the measured electrical conductivity using a conversion coefficient. This coefficient is between 0.13 -0.17 dS/m for 1 M (moles/L) of ionic strength [4]. Assuming the conversion coefficient of 0.16, for the conductivity of our experimental media σ = 0.1188 S/m, the corresponding ionic strength is 0.19 M or 190 mol/m 3 . Under these conditions, the calculated Debye length is 0.7 nm, see calculations in S8.

Comment on electrolysis in experiments
One of the major limitations of the current approach is the formation of bubbles caused by electrolysis of the buffer, which ultimately limited the number of electric pulses that could be delivered and the length of time that the MTs could be exposed. This limitation could be solved in future work by using a fully throughflow microfluidic channel connected to external reservoirs with degassed buffers. In such a way, the buffer could be continuously replaced, removing the gas 4 being formed. However, another solution that could be used to prevent the formation of bubbles is to avoid the charge transfer between the electrodes and the buffer. This could be achieved by insulating the electrodes and delivering the electric field to the channel via capacitive coupling, which would be most effective for pulses with nanosecond-scale duration or shorter.