Zepto

The flat and nanofaceted Al₂O₃ substrates were cleaned through incubation in 2% Hellmanex solution (Hellma GmbH & Co. KG, Müllheim, Germany) for 1 h at room temperature, washing with HPLC-grade water (Carl Roth GmbH, Karlsruhe, Germany), and drying in a stream of argon. Finally, the substrates were treated for 30 s with an oxygen plasma (diener Zepto, Diener electronic, Ebhausen, Germany) to remove any remaining organic contaminations.
Ferritin from equine spleen (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was prepared at concentrations of 10 mg/mL and 30 mg/mL in PBS (pH 7.4, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Furthermore, 200 µL of the prepared protein solution was carefully placed on each substrate surface and incubated for 5 h at room temperature in an environmental chamber. After incubation, the substrates were rinsed with 1 mL of HPLC-grade water and dried in a stream of argon.

The epi-ready, p-doped Si(100) wafers employed as substrates were purchased from Siegert Wafer (Aachen, Germany). The wafers were cut into 1 × 1 cm² pieces and cleaned for 15 min at 75 °C in fresh RCA1 solution (1:1:5 NH₄OH:H₂O₂:H₂O). Ion beam nanopatterning was performed as previously described [15 (link)] using a Kaufman-type KDC 40 ion source (Kaufman & Robinson Inc., Fort Collins, CO, USA) mounted in a vacuum chamber with a base pressure of about 1 × 10⁻⁷ mbar. Irradiation was performed with 500 eV Ar⁺ at an incident angle of 67° with respect to the surface normal. The applied flux and fluence were 2 × 10¹⁴ cm⁻²s⁻¹ and 1 × 10¹⁷ cm⁻², respectively. Immediately before incubation with hIAPP, all substrates were treated with oxygen plasma (diener Zepto, Diener electronic, Ebhausen, Germany) for 30 s to achieve a reproducible highly oxidized surface chemical state.

The microfluidic structures were produced by soft lithography [35 (link)]. PDMS was poured on a master, which was the negative of the microfluidic-channel network. The master was developed from a photolithographically exposed negative resist. Depending on the structure size, different resist systems can be used. The dry film resist (Elga Europe s.r.l, Milan, Italy) is suitable for structures down to the lower micrometer range. For sub-micrometer feature sizes, SU-8 (NanoTM MicroChem, Kayaku, Westborough, MA, USA) can be used. Next, PDMS was poured over the master and baked for 12 h at 40 °C (lower temperatures are preferred to reduce shrinking) [36 (link)]. Then, the PDMS-chip was removed from the master and plasma-bonded to a glass slide. For this, we used a low-pressure plasma cleaner (Zepto, Diener Electronic GmbH & Co. KG, Ebhausen, Germany). Oxygen plasma treatment was done for 45 s at medium vacuum (<102 Pa) with an oxygen influx of 80 l_n/h and 15 W output power at 40 kHz frequency.
For the fabrication of hydrogel-based microsystems, two large-scale integration concepts are established: the flip-chip technology and in situ polymerization [37 (link),38 (link)]. An overview of the procedures is given in the illustrations of Figure 3. On the left, the process of the flip-chip method is illustrated, and, on the right-hand side, in situ polymerization is illustrated.

Acid-washed glass coverslips (HCO₃ (20%); 1 h at RT) were plasma cleaned (Zepto; Diener Electronic GmbH). Plastic channels ((sticky slides IV0.4, Ibidi) were attached to the coverslips and subsequently coated (30 min at RT) with PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-RGD (1 mg/ml in PBS, SuSoS) to generate stabile, covalently bound RGD-containing substrates. Fragile RGD substrates were generated by introducing a biotin-neutravidin–biotin bridge linking RGD to the PLL–PEG (poly-l-lysine-PEG) backbone. The biotin–avidin bond ruptures at forces >160pN^{84 (link)}, thus allowing platelets to mechanically remove an RGD-containing ligand when integrin pulling forces overcome 160 pN. Plasma-cleaned glass coverslips were coated with PLL(20)-g[3.5]–PEG(2)/PEG(3.4)–biotin (50%) (0.1 mg/ml diluted in 1 mg/ml PLL(20)-g[3.5]–PEG(2)/PEG(3.4) in PBS; 30 min at RT; SuSoS). PLL-g-PEG-biotin-functionalized coverslips were subsequently incubated with NeutrAvidin-FITC (200 µg/ml in PBS, Invitrogen) for 30 min at RT, followed by the incubation with cyclo RGDfk-biotin [Arg-Gly-Asp-d-Phe-Lys(Biotin-PEG-PEG)] (0.1 μM in ddH₂O, Peptides International) for 30 min at RT. Slides were washed with PBS and incubated with washed platelets in the presence of platelet activators (4 µM ADP, 2 µM U46619) and 1 mM Ca²⁺/Mg²⁺. Time-lapse movies were recorded as described above.

In the case of upright STED imaging, soda lime glass (microscope slides DIN ISO 8037-1, Epredia) was activated by an oxygen plasma [p(O₂) = 0.2 mbar, E_output = 13.7 J, t = 3 s, d = 9.5 cm] (Plasma cleaner Zepto, Diener electronic GmbH + Co.KG)^{29 (link)}. In the case of inverse STED imaging, borosilicate glass was coated with 30 nm of SiO (UNIVEX400, Leybold)^{38 (link)}. Hot water incubation was performed at 80 °C for 30 min. GPMVs were spread immediately after incubation to form SPMBs^{29 (link)}.

For
the introduction of oxygen-containing groups at the surface of the
fibers, eFMs were activated by oxygen plasma as described elsewhere.^{29 (link)} Briefly, the PCL eFMs were placed in the chamber
of a low-pressure plasma system (Zepto, Diener Electronic), subjected
to vacuum (<0.4 mbar), and then filled with oxygen. The pressure
of the plasma chamber was maintained near 1 mbar during the treatment.
A power intensity of 30 W was applied for 5 min.

Carbon-coated copper grids, 200 mesh (Science Services, Munich, Germany) were treated with oxygen plasma (Zepto, Diener electronic GmbH, Ebhausen, Germany). After this, 3 µL of precipitation-purified rAAV sample [25 (link)] was applied to the grid and incubated for 2 min. Excess liquid was drained off, the grid was dried at room temperature and washed with three drops of distilled water. Negative staining was performed using 3 µL 2% (v/v) uranyl acetate replacement stain (Science Services) for 30 s. Excess liquid was drained off and grids were dried before channeling the sample into the microscope. rAAVs were visualized with a CM100 (PW6021) instrument (Philips, Hamburg, Germany) with an acceleration voltage of 80 kV. Images were analyzed using the Soft Imaging Viewer (Olympus, Münster, Germany) and ImageJ [50 (link)].