Spectramax m5e multi mode microplate reader

The protein buffers were exchanged for anisotropy buffer (20 mM Hepes/KOH pH 7.5, 200 mM KCl, 5 mM MgCl₂, 1 mM DTT) using Zeba spin columns (Thermo Scientific). 1 µM BSA was added to prevent unspecific binding of the fluorescently labelled peptide to the microplate (Greiner Bio-One opaque 384 well plate). Fluorescence anisotropy measurements were performed using the LP-peptide (MFSLPTL) or the NR-peptide (NRLLLTG) labelled at the N terminus with fluorescein (Peptide Specialty Laboratories GmbH, Heidelberg). Two-fold serial dilutions of each protein (ScSsz1-Zuo1N or ScSsz1-Zuo1NΔLP) were mixed in a 1:1 ratio with 40 nM peptides in anisotropy buffer in 384-well opaque plates (Greiner Bio One) and were incubated at room temperature for 30 min. Measurements were performed in triplicate using a plate reader (SpectraMax M5e Multi Mode Microplate reader, Molecular Devices). To determine the binding constant (K_D) the data were fitted to a one-site binding equation using Python⁵⁷ .

An ELISA kit was used based on the protocol provided by the manufacturer (Sino Biological, Inc. Beijing, China). For the proof‐of‐concept of S100A1 extraction from the skin model, 100 µL of sample from 150 µL total volume after elution was added to the microplate's pre‐coated wells. While for the establishment of the ELISA standard curve of S100A1 extracted using a microneedle patch, the eluted sample from microneedles was diluted 30 times (5 µL eluted sample in 150 µL sample dilution buffer) and 100 µL of diluted samples were added into the microplate wells. For mouse serum detection, the serum was diluted 30 times first and 100 µL of diluted samples were added into the microwells for measurement. For the mouse ISF detection, the eluted ISF from microneedles was diluted 30 times and 100 µL of diluted samples were added into the microwells for measurement. After the sample was added and reacted for 2 h, the microplate was washed with washing buffer three times, and the detection antibody with HRP conjugation was added and reacted for 1 h. After finishing another washing cycle, 200 µL of TMB substrate solution was added to each well and incubated at room temperature in the dark for 15 min before adding 50 µL of stop solution to each well and measured using a microplate reader (SpectraMax M5e Multi‐Mode Microplate Reader, Molecular Devices) at 450 nm after the reaction stopped.

hex4_A5U oligonucleotides were labeled with either 5′ 6-carboxyfluorescein (hex4_A5U-5F) or 3′ carboxytetramethylrhodamine (hex4_A5U-3T) or both fluorophores (hex4_A5U-5F3T). Reactions were set up in triplicate in 96-well Nunclon plates (Thermo Fisher Scientific, Waltham, MA) containing 200 nM of either hex4_A5U-5F3T or a mix of 100 nM hex4_A5U-5F + 100 nM hex4_A5U-3T annealed in 10 mM TBA (pH 7.5) + 100 mM KCl or 100 mM LiCl. Protein was added at 0, 200 nM, 500 nM, or 1000 nM concentrations and incubated for 1 h at 4 °C before data collection. Data were collected on a Spectramax M5^e Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and processing was performed as previously described [33 (link)]. Labeling and data collection for A5U^R20 oligonucleotides were done as described for hex4_A5U.

We prepared another 3 groups of experimental rats following the same experimental procedures to measure the permeability of the BBB using the Evans blue assay as previously described [18 (link)]. Evans blue solution (Sigma) at a concentration of 2% at 5 ml/kg body weight was injected into the femoral vein 1 h before the rats were euthanized. The circulating dye was cleared with a perfusion of cold PBS. The harvested brain tissue was homogenized and incubated in dicarboxamide at 37 °C for 48 h. After centrifugation at 300×g for 5 min, the optical density (OD) of the supernatants was measured at 620 nm absorbance with a SpectraMax M5e Multi-Mode Microplate Reader (Molecular Devices).