For design simplicity and cost control it would be advantageous to have a single generic MEMS-based biosensing core that could be easily modified to produce a family of biosensors each with a unique specificity. With that in mind, we have chosen to work on the development of a generic MEMS device that will detect an internal change in viscosity. In this model system, the sensor actually is a cantilever-based viscometer. We successfully have constructed a microviscometer sensing unit (Fig 2) by adapting a system developed by Jeckelmann and Seibold(14) for monitoring the blood glucose levels of people with diabetes. In their system (non-MEMS) changes in viscosity are measured based on competition between glucose and dextran (a glucose polymer) for binding with the quadrivalent concanavalin A (conA) molecule. In the absence of free glucose, four large dextran molecules will bind to each molecule of ConA producing a high viscosity gel-like matrix. If glucose enters the system, then it will compete for the ConA binding sites resulting in a drop in viscosity. The MEMS-type viscosity biosensor that we developed works by measuring the deflection of a cantilever in inverse proportion to the viscosity (Fig 3). Therefore, the greater the deflection of the cantilever, the lower the viscosity and the greater the signal recorded by the system. For the device we are designing, enzymatic glucose production (generated from a polysaccharide) will be the signal that the TRAP-based biosensor has been triggered by RAP binding.
As mentioned above, in an effort to circumvent the universal problem of biofouling that befalls all implantable biosensors, we have developed a strategy in which we will not attempt to pass the ligand to be detected (RAP) into the biosensor. Instead we will attempt to transduce a signal into the biosensor using a transmembrane-based conformational protein switch, in essence mimicking the natural process by which most cells receive information about their environment. To accomplish this we are taking a protein-engineering approach to the problem in which the extracellular domain of TRAP will be fused to a transmembrane conformational switching domain that in turn is fused to a glucosidase. When the hybrid TRAP molecule is unbound, the glucosidase on the interior of the biosensor will be in an inactive conformation and the biosensor will remain in high viscosity status, indicating that there are no bacteria nearby. However, if staphylococcal produced RAP is present near the joint, it will bind to the engineered TRAP proteins on the surface of the biosensor and the molecular switch will trigger activation of the glucosidase moiety. The activated glucosidase will release free glucose from a substrate glucose polymer which will, in turn, competitively bind to the concanavalin A, displacing the dextran and resulting in a decrease in viscosity which will initiate release of the multicomponent treatment regimen described above.
A similar protein engineering approach will be used to monitor the concentration of the released pharmacological reagents. For each ligand to be monitored, a hybrid receptor-switch-glucosidase protein will be engineered to function as a “front end” for the MEMS glucose-based viscometer. Therefore the most difficult developmental process associated with this project will serve multiple masters.