They used FectoPRO to transfect plasmid DNA in HEK293F for recombinant (scFv)2-Fc and (VHH)2-Fc Antibody production. HEK293F cells were cultured in suspension in FreestyleTM 293 expression medium at 37 °C, 110 rpm, 8% CO2, and 95% humidity. Cells were sub-cultured 3× per week at 0.3–0.5 million/mL in culture flasks. For counting and viability checks, an automated cell counter was used, and cells were diluted 1:1 in 0.4% Trypan Blue. Plasmids encoding for the recombinant antibodies were amplified in chemically competent E. coli XL1-blue bacteria and harvested using Mini/Midi/Maxi prep. HEK293F cells (1 million/mL) were transfected with the FectoPRO using 500 μg DNA/million cells for the (scFv)2-/(VHH)2-Fc (IgG1) format. After 2.5 h, transfection was boosted by the addition of 2 mM sodium butyrate. Cells were incubated at 37 °C, 110 rpm, 8% CO2, and 95% humidity. After 4–6 days, the cells were checked for viability, and the antibody was harvested when the viability dropped below 60%, as detected using automated cell counting. To harvest the antibody, the culture was first centrifuged at 300g for 10 min at 4 °C to remove the cells, and then at 4816g for 1 h at 4 °C to remove cell debris and aggregates. The supernatant, containing the recombinant antibodies in culture medium, was stored at 4 °C until further use. Antibody concentrations were assessed using Octet measurements with a Protein A-coated sensor and a hIgG1 standard curve.

Industrial food processes are monitored to ensure that food is being produced with good quality, yield, and productivity. For developing innovative real-time monitoring and control strategies, real-time sensors are needed that can continuously report chemical and biochemical data of the manufacturing process. Here, we describe a generalizable methodology to develop affinity-based biosensors for the continuous monitoring of small molecules in industrial food processes. Phage-display antibody fragments were developed for the measurement of small molecules, as exemplified with the measurement of glycoalkaloids (GAs) in potato fruit juice. The recombinant antibodies were selected for use in a competition-based biosensor with single-molecule resolution, called biosensing by particle motion, using assay architectures with free particles as well as tethered particles. The resulting sensor measures GAs in the micromolar range, is reversible, has a measurement response time below 5 min, and enables continuous monitoring of GAs in protein-rich solutions for more than 20 h with concentration measurement errors below 15%. The demonstrated biosensor gives the perspective to enable a variety of monitoring and control strategies based on continuous measurement of small molecules in industrial food processes.