N n carbonyldiimidazole cdi

pKL immobilization onto EG4-SAMs proceeded via a two steps reaction (Figure S3—Supporting Information): (a) activation of -OH groups of EG4-SAMs using N,N’-Carbonyldiimidazole (CDI; Sigma-Aldrich, St. Louis, MO, USA) to create reactive imidazolyl-carbamate groups (CDI activation); (b) pKL covalent immobilization on activated SAMs through its free NH₂ groups (pKL immobilization).
To prepare EG4-SAMs with different degrees of activation, SAMs were immersed in solutions with different CDI concentrations: 0, 0.3 and 30 mg/mL, in anhydrous tetrahydrofuran (THF; Merck) as described elsewhere [24 (link)]. Then, CDI-activated EG4-SAMs were immersed in a pKL solution (0.5 mg/mL) prepared in dimethyl sulfoxide (DMSO; BDH Prolabo) with 0.02% (v/v) triethylamine (Sigma-Aldrich). Samples were incubated at 40 °C, 100 rotations per minute (rpm) agitation for 24 h. Control surfaces were prepared by incubating CDI-activated EG4-SAMs in a DMSO solution with 0.02% triethylamine (buffer-SAMs).
Afterwards, pKL-SAMs and buffer-SAMs were rinsed with DMSO (three times, during 5 min in an ultrasonic bath) and Type I water (Milli-Q^® ultrapure water: Resistivity > 18 MΩ/cm; Conductivity < 0.056 µS/cm; Total organic carbons < 50 ppb) (twice for 5 min in an ultrasonic bath). Finally, samples were dried with an argon stream and stored under an inert atmosphere until further use.

Authentic Kwa (≥96%) was isolated from the tuberous roots of P. candollei using the method described in a previous report [15 (link)]. OVA (≥98%), BSA (≥97%), human serum albumin (HSA, ≥99%), and N,N′-carbonyldiimidazole (CDI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A previously reported anti-glycyrrhizin (GC) monoclonal antibody (MAb 2H2) [26 (link)] and anti-harringtonine (HT) MAb (MAb 1D2) [27 (link)] were used as candidate proteins for bioimprinting. Anti-OVA antibody (6C8) (ab17293), anti-mouse immunoglobulin G1 (IgG1) goat antibody (horseradish peroxidase, HRP) (ab97240), anti-mouse IgG goat antibody (HRP) (ab6789), and goat F(ab) anti-mouse IgG H&L (HRP) (ab6823) were obtained from Abcam (Cambridge, MA, USA). Glutaraldehyde, used for crosslinking denatured proteins, and an ELISA POD Substrate 3,3,5,5-tetramethylbenzidine (TMB) Kit, used as a substrate solution for ELISA and ELBIA, were obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals were standard commercial products of analytical grade.
Sample preparation for the analysis of Kwa is described in the Supplementary Materials.