Dynabeads m 270 amine

N^α-(tert-butoxycarbonyl)-L-Lys (Boc-Lys), ε-aminocaproic acid (6-ACA), AA, FA, BSA, trifluoroacetic acid, 3,3′,5,5′-tetramethylbenzidine (TMB), human MPO, and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO). Malondialdehyde bis(dimethyl) acetal, Dynabeads M-270 Amine, CarboxyLink Kit, the Imject EDC mcKLH Spin Kit, goat anti-rabbit IgG (H + L) antibody with HRP, goat anti-human IgG/IgM secondary antibody, and goat anti-mouse IgM secondary antibody were obtained from Thermo Scientific (Rockford, IL). Protein A column and autoLDL™ Cholesterol kit were purchased from GE Healthcare Life Sciences (Pittsburgh, PA) and Pointe Scientific (Lincoln Park, MI), respectively. EnVision + Single Reagents anti-mouse/rabbit-HRP were from Dako North America, Inc. (Carpentaria, CA). The anti-M2AA mouse monoclonal antibody (1F83)²⁴ and rabbit polyclonal antibody^{48 (link)} were provided by Drs. Koji Uchida (Nagoya University) and Todd A. Wyatt (University of Nebraska), respectively.

The ssDNA sequences of aptamers FB1_39 [25 (link)], FB1_39t3 [26 (link)], FB1_10 [27 (link)], AF_AB3 [20 (link)] and AF_APT1 [19 (link)], and all aptamers modified with 5′-6-carboxyfluorescein were purchased from Bio-Fab Research Srl (Rome, Italy) as lyophilised form and with HPLC purity. Then, each aptamer was solubilized in the respective buffer solution. The ssDNA aptamer sequences for FB1 and AFB1 and the relevant composition of buffer solutions are reported in Table 1. All chemicals for buffer solutions and all other purposes were purchased from Sigma-Aldrich (Milan, Italy). The Dynabeads™ M-270 Amine and the DynaMag™-2 Magnet were obtained from Invitrogen (Life Technologies, Monza, Italy). FB1 and AFB1 standards were purchased from Vinci-Biochem Srl (Florence, Italy).
Mycotoxins stock solutions were prepared by dissolving AFB1 powder in toluene:acetonitrile (90:10, v/v) at concentration of 0.5 mg/mL, FB1 powder in acetonitrile:water (1;1, v/v) at concentration of 1 mg/mL and OTA powder in toluene:acetic acid (99:1; v/v) at concentration of 0.5 mg/mL. Mycotoxins working solutions were prepared by drying appropriate aliquots of stock standard solutions and redissolving them in the respective buffer solutions at concentrations ranging from 0.1 nM to 200 µM. Information relevant to OTA aptamers and relative standard preparation are reported elsewhere [31 (link),39 (link)].

1 mL of 10
μM dimeric protein Gx with a C-terminal cysteine or protein
M variant in PBS was incubated with 5 mM TCEP for 1 h at RT. The protein
was desalted on a PD-10 column equilibrated in 100 mM sodium phosphate
pH 7.0. 4x 150 μL of 1 μM cetuximab, 1 μM protein
Gx in 100 mM sodium phosphate pH 7.0 was prepared and protein Gx was
photo-cross-linked to cetuximab using a reported procedure.^{1 (link)} 0.6 mg of superparamagnetic beads (Dynabeads
M-270 amine, Invitrogen) were equilibrated in PBS pH 7.2 and labeled
with 1.8 μg sulfo-SMCC in PBS pH 7.2, 50% v/v DMSO, 20 μL
for 30 min at RT under slow tilt and rotation. The beads were washed
twice with 200 μL of 100 mM sodium phosphate at pH 7.0 and 25
μM TCEP. The beads were resuspended with the photo-cross-linked
cetuximab or protein M variant and incubated for 3 h at RT with slow
tilt and rotation. The coupling efficiency was estimated by measuring
the absorbance at 280 nm before and after coupling. After the coupling,
the beads were washed 2× with 400 μL of storage buffer
(PBS + 0.01% Tween-20) and stored in 100 μL of storage buffer
at 4 °C.

2-Ethyl-1-hexanol (98%, Sigma); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (Carbosynth); 1,6-diaminohexane (98%, Sigma); PEG-bis-(N-succinimidyl succinate) (M_w = 2,000, Sigma); DyLight 405 NHS ester (ThermoFisher); FITC NHS ester (ThermoFisher); BSA (heat-shock fraction, pH 7.0, ≥98%; Sigma); streptavidin from Streptomyces avidinii (Sigma); Tamavidin 2-HOT, recombinant (Wako Chemicals); Dynabeads M-270 amine (Invitrogen); Dynabeads MyOne carboxylic acid (Invitrogen); 1 M MgCl₂ (Invitrogen); 1 M Tris pH 8.0 RNase free (Invitrogen); 5 M NaCl (Invitrogen); EvaGreen (Biotium); KAPA HiFi HotStart PCR kit (Roche); Micellula DNA emulsion and purification kit (EURx); ibidi anti-evaporation oil (ibidi); 30% (19:1 monomer:bis) acrylamide solution (Bio-Rad); and SYBR Gold (ThermoFisher) were used as received. All the other chemicals used were purchased from Sigma. The enzymes were purchased from New England Biolabs, unless noted otherwise.

Dynabeads functionalized with amine groups (DB-NH₂, Dynabeads^TM M-270 Amine) and Apolipoprotein B Monoclonal Antibody (LDL-apoB) were obtained from Invitrogen (Poland). Malondialdehyde ApoB-100 Monoclonal Antibody (MDA-LDL-apoB) was purchased from MyBioSource, Inc. (San Diego, CA, USA). Malondialdehyde-Modified LDL (MDA-LDL) was obtained from Cell Biolabs, Inc. (San Diego, CA, USA). Ferrocene Carboxylic N-hydroxysuccinimide Ester (Fc-NHS) was acquired from Fivephoton Biochemicals (San Diego, CA, USA). N-Hydroxysuccinimide (NHS), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), Anthraquinone-2-carboxylic acid (AQ-COOH), Na₂SO₄, trizma base (Tris), 2-(N-Morpholino)ethanesulfonic acid hydrate (MES), dimethyl sulfoxide (DMSO), low density lipoprotein (LDL) and humans serum albumin (HSA) were purchased from Sigma-Aldrich (Poznań, Poland). High density lipoprotein (HDL) was bought from EMD Millipore Corporation (Burlington, MA, USA). Ethanol and methanol were purchased from POCH (Gliwice, Poland). Deionized water (resistivity of 18.2 MΩ cm; Millipore Mili-Q, Bedford, MA, USA) was used for preparation of all aqueous solutions. Human blood serum was obtained from Sigma-Aldrich.