Real-Time Immunosensor for Small-Molecule Monitoring in Industrial Food Processes

Abstract

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.

Molecular Origins of Long-Term Changes in a Competitive Continuous Biosensor with Single-Molecule Resolution

“…It is important to develop diverse research methodologies to quantify long-term changes and unravel their molecular origins because long-term operation is an important aim for continuous biosensors. ,,,− The methodologies in this article were studied on a competition-based small-molecule BPM sensor. We think that the methodologies can also be applied to other affinity-based sensors, especially the single-sided aging experiments and the equilibrium-shift experiments. ,− , The experiments of this paper were performed using buffer solutions; as a next step, it will be relevant to study how long-term changes are affected by the composition of the fluid. Also, transport properties need to be considered; analyte transport in a continuous sensor is driven by advection and/or diffusion, so it is relevant to study how these mechanisms affect the long-term changes of the biosensor.…”

“…We think that the methodologies can also be applied to other affinity-based sensors, especially the single-sided aging experiments and the equilibrium-shift experiments. 6,[10][11][12][13][14][15][16]18 The experiments of this paper were performed using buffer solutions; as a next step, it will be relevant to study how long-term changes are affected by the composition of the fluid. Also, transport properties need to be considered; analyte transport in a continuous sensor is driven by advection and/or diffusion, 32 so it is relevant to study how these mechanisms affect the longterm changes of the biosensor.…”

“…Biosensing technologies for the continuous monitoring of biomolecules are being developed to enable monitoring and control functionalities in fields such as healthcare, , industrial biotechnology, organ-on-chip research, , industrial food processing, , and environmental monitoring . Continuous sensors are commercially available for the continuous minimally invasive monitoring of glucose .…”

“…The particles switch between unbound states with high mobility and bound states with low mobility, due to affinity-based single-molecule interactions that are influenced by analyte molecules. ,,, The particle switching rate depends on the analyte concentration in solution and can be continuously measured using video microscopy. ,, The sensing method does not consume or produce any reagents, which makes it suitable for long-term continuous sensing applications. In previous studies, BPM sensors functioned up to 24 h, and mathematical corrections were applied to compensate for signal changes over time. ,, However, the origins of signal changes were not clear.…”

Integrated sampling-and-sensing using microdialysis and biosensing by particle motion for continuous cortisol monitoring

“…The particle motion in a field of view of 1129 × 706 μm was recorded for at least 10 min at a frame rate of 30 Hz with 5 ms exposure time under dark-field illumination conditions with real-time particle tracking software as described by Bergkamp et al 35 The localization of particles, analysis of particle motion and detection of switching events were done using phasor-based localization 36 and Maximum-likelihood Multiple-windows Change Point Detection method (MM-CPD). 35 The sensor response was corrected for drift over time using the method as described by Vu et al 37 Image recording and data analysis of azorubine. Glass slides (25 × 75 mm) were functionalized with a custommade 12 μl flow cell sticker and connected to flexible tubing and the microdialysis probe, similar as described for BPM experiments.…”