Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media
Abstract
:1. Introduction
2. Results and Discussion
- (i)
- When the two cation binding sites S1 and S2 are occupied by ions, the protein undergoes a conformational change that involves the isomerization of a non-proline peptide bond from trans- to its cis-conformation. The isomerization presents a certain energy barrier that needs to be overcome. This change has been called “locking” and it largely increases ConA’s affinity to the bound ions. It has been shown that only locked ConA can bind to carbohydrates, but also metal-free ConA can be in its locked/native state. Incubating with excess carbohydrates results in a shift of unlocked/locked equilibrium of metal free ConA from around 12% to over 60% of the locked conformation in an experimental set up. Varying the ion species in S1 and S2 has shown little change in binding strength to the ligand. However, the most important divalent metal cation Ca2+ is missing in the growth medium, and even though no direct explanations can be found in the literature, it is evident that ConA is a very cation sensitive protein with three cation binding sites, and therefore variations in ligand binding strength due to changes in specific cation concentrations cannot be ruled out.
- (ii)
- We also considered the phosphorylation of glucosyl residues that could lead to covalent bonds between dextran molecules, which would lead to a permanent increase in viscosity by strengthening the molecular network of the assays. In the lab, phosphorylation of dextran was achieved under exclusion of water at high temperatures (90 and 120 °C, respectively for 6 h, only approximately 10%) and low pH [38,39]. Therefore, phosphorylation appears rather unlikely to happen spontaneously under the conditions present in the sensor cavity.
- (iii)
- Some of the salts used in Noack medium are applied in protein crystallization protocols, namely NH4+, SO4+, and phosphate. Although their concentration in the medium is much smaller, the ions mentioned belong to the most effective salting-out agents of the Hofmeister series [40]. Thus, it might be expected that aggregation of ConA to higher mers than tetramers strengthens the intermolecular network between assay components and causes a higher viscosity. This assumption is supported by the fact that the applied concentration of ConA is close to its solubility limit in solutions of electrolytes used in the medium [41].
- (iv)
- Agglomeration induced by electrolytes and the associated increase in viscosity were already described by Kim and Myerson for lysozyme [42]. Concentrations of the involved ions and the apparent time constants were of the same order of magnitude as observed in this work. Their study also pointed to the electro-viscous effect that accounts for the property of proteins to surround themselves with a cloud of dissolved ions. The cloud composition, which depends on ionic species and concentrations, determines the forces necessary to move the protein molecule within the solvent, and thus the viscosity of the solution.
3. Materials and Methods
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ConA | Concanavalin A |
MEMS | Microelectromechanical system |
OD600 | Optical density at 600 nm |
References
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Standard Electrolyte SEL | Mineral Salt Medium [37] | ||||
---|---|---|---|---|---|
Component | g·L−1 | mM | Component | g·L−1 | mM |
Tris/HCl | 1.21 | 7.7 | KH2PO4 | 2.72 | 20 |
NaCl | 7.76 | 133 | Na2HPO4 × 2H2O | 3.56 | 20 |
MnSO4 × H2O | 0.169 | 1.00 | NaCl | 5.00 | 85.6 |
CaCl2 | 0.147 | 1.32 | Na2SO4 | 1.10 | 7.74 |
NaN3 | 0.5 | 7.7 | NH4Cl | 0.50 | 9.35 |
MgCl2 × 6H2O | 0.04 | 0.197 | |||
Affinity assay = SEL + ConA + dextran | FeCl3 × 6H2O | 0.008 | 0.030 | ||
Dextran T2000 | 70 | 0.035 | MnSO4 × H2O | 0.004 | 0.024 |
ConA | 7 | 0.264 | Thiamine | 0.05 | 0.148 |
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Theuer, L.; Lehmann, M.; Junne, S.; Neubauer, P.; Birkholz, M. Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media. Int. J. Mol. Sci. 2017, 18, 1235. https://doi.org/10.3390/ijms18061235
Theuer L, Lehmann M, Junne S, Neubauer P, Birkholz M. Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media. International Journal of Molecular Sciences. 2017; 18(6):1235. https://doi.org/10.3390/ijms18061235
Chicago/Turabian StyleTheuer, Lorenz, Micha Lehmann, Stefan Junne, Peter Neubauer, and Mario Birkholz. 2017. "Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media" International Journal of Molecular Sciences 18, no. 6: 1235. https://doi.org/10.3390/ijms18061235
APA StyleTheuer, L., Lehmann, M., Junne, S., Neubauer, P., & Birkholz, M. (2017). Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media. International Journal of Molecular Sciences, 18(6), 1235. https://doi.org/10.3390/ijms18061235