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Protein Engineering for Sensors & Bioelectronics

Cambridge Analytical Biotechnology

Even though protein based biosensors have been successfully developed, proteins have not evolved with the primary selection goal of desired analyte specificity coupled with a transducer mechanism suitable for biosensor integration. Similarly, the hunt for naturally occurring bioelectronic components does not reveal structures optimised for assembly and deployment in circuitry. A powerful tool currently being used to expand the family of biomaterials is protein engineering.

The vision converges towards modular molecular-engineering in which integrated signal transduction function and binding site characteristics are engineered in tandem with self assembling nanostructures producing auto-immobilisation and stabilisation of the proteins.

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Star assembly of Enginerred Red Fluorescent Protein

Three prevailing methods in protein engineering exist; rational protein design, directed evolution and de novo design. The first two methods have become some of the most powerful and widely used tools in enzyme biotechnology.
If the protein is to be designed for electrochemical or optical measurements, then understanding the principle of electron transfer (ET) or light harvesting or emission in proteins is important in finding strategies for facilitating communication between proteins and their surrounding environment.

For example, one important factor that influences the electron transfer rate is the distance between the redox site and the electrode. Direct electron transfer between protein and an electrode is controlled mainly by three factors:
• reorganisation energies
• potential differences and orientations of the redox-active sites involved in each oxidation state
• distances between redox-active sites and charge transfer agent/mediator.

In proteins, the electron transfer rates drop by 104 when the distance between an electron donor and acceptor is increased from 8 to 17Å. Analogies can be gained from examination of an optical Förster Resonance Energy Transfer system where distance between acceptor and donor determine the fluorescence coupling efficiency. Consideration and understanding of these processes provides the knowledge to photon activated electron transfer; showing dual protein transduction coupling; having enhanced fluorescence etc.

Furthermore, one of the main problems of many biosensors and bioelectronic components is that they lack intrinsic stability. In some notable cases this has not been a drawback, allowing for example, the successful commercialization of glucose biosensors world-wide. However, the majority of enzymes and proteins, with useful functionality, are labile and require stabilisation to produce viable devices. A major breakthrough is the design of self-assembling protein structures to improve stability and offer new nanostructured materials