Electrochemical Detection of Insulin with a Novel Protein-Based Artificial Receptor

Abstract

In this paper, we have developed a new electrochemical method to detect insulin with a protein-based artificial receptor. Diabetes is one of the most serious diseases all over the world. For the treatment of diabetes and maintenance of the patient's quality of life, the control of blood glucose level is important. Usually the blood glucose level in human body is regulated by insulin. Therefore, it is very important to know the status of both glucose and insulin in blood for the treatment of diabetes. Currently, it has become possible to measure the blood glucose level by patients themselves with bioelectrochemical sensors, however, there is no convenient method to measure insulin by patients themselves. In recent years, antibody-based and aptamer-based electrochemical insulin sensors have been developed. Although these sensors are highly sensitive, they may also detect physiologically inactive insulin such as partially digested or mutation insulin because the insulin recognition molecules of these sensors specifically recognize a part of insulin. However, it is desired to detect only active insulin for accurate evaluation of insulin which is functional in blood. Therefore, it is necessary to develop a novel sensor based on a new target recognition mechanism for the accurate evaluation of insulin. In our strategy, we utilized an insulin receptor, because the recognition by the insulin receptor is one of the essential processes for insulin to exhibit its functional role in blood. It has been reported that two domains in the insulin receptor, αCT segment and L1CR domain, recognize an insulin molecule by sandwiching format. However, the insulin receptor is typically buried in a cellular membrane and thus it is difficult to use the receptor with a native format for electrochemical detection. Hence, we have developed a novel artificial insulin receptor by connecting these two domains with a linker. We considered that this new artificial insulin receptor alters its structure in response to insulin recognition, and thus electrochemical properties of an electrode modified with the artificial receptor should reflect the structural change. In order to modify the electrode, we utilized three-time-repeats of a gold-binding peptide (GBP) that is known to assemble on a gold surface spontaneously. These domains were genetically fused and resulting artificial insulin receptors were termed as 3GαL and αL3G depending on their sequence of the domains, respectively. The novel artificial receptors were expressed by introducing plasmids encoding each recombinant gene to animal cells, then purified utilizing histidine-tag affinity. The expression and purification of each receptor was confirmed by SDS-PAGE. The ability of self-assembling on a gold surface was evaluated by QCM-D. The artificial receptor modified electrode was prepared by placing each protein solution on the gold surface of electrodes. The electrochemical response of the modified electrode to insulin was evaluated using electrochemical impedance spectroscopy (EIS). EIS measurement was performed in buffer solution in the presence of 5 mM [Fe(CN)6]3-/4- (the frequency range: 100 mHz to 100 kHz, potential: REST, amplitude: 10 mV). The comparisons of Nyquist plots revealed that the diameters of semicircle increased in the case of electrodes modified with the artificial receptors, when compared with an unmodified electrode. This is probably because the artificial receptors are proteins which inhibit interfacial electron transfer. When insulin was added to the solution, the diameters of semicircle further increased, while no drastic increase was observed when C-peptide was added. Comparison of the responses between electrodes modified with 3GαL and αL3G revealed that the electrode modified with 3GαL exhibited a better S/N ratio in response to insulin. In case of 3GαL, the side of αCT segment was immobilized on the gold surface and L1CR domain whose size is much larger than αCT segment kept rather free. In contrast, in case of αL3G, the side of larger domain of L1CR was already fixed and the smaller part of αCT segment kept rather free. Therefore, it was considered that the effect of structural change induced by the recognition of insulin on the electrochemical property of electrode should be more significant in the case of 3GαL than the case of αL3G. From these results, it has been concluded that insulin can be measured electrochemically with the electrode modified with the protein-based artificial receptors.