On the Design of Low-Cost Fluorescent Protein Biosensors

Abstract

Proteins are the cell’s workhorses as they are involved in essentially all cellular activities. From the birth of a daughter cell until its death during apoptosis, proteins perform various functions as needed. Such versatility can be attributed to the seemingly infinite ways that the amino acids are sequenced in a polypeptide. The polypeptide backbone and each amino acid functional group contribute unique intermolecular interactions that determine the protein’s shape and size. However, more importantly, these interactions determine protein function, that is, its ability to interact with the external environment. For the same reasons, proteins make ideal biorecognition elements for sensors (see below and Fig. 1). Protein enzymes and receptors developed unmatched sensitivity and selectivity for their substrates through millions of years of natural selection. In contrast, chemically synthesized artificial receptors seldom approach the same level of sophistication in ligand selectivity, sensitivity range and ease of production. In addition, there is available to the sensor researcher an ever expanding library of proteins for various ligands important in all manner of application such as disease biomarkers, toxins, contaminants, drugs, etc. Using tools of genetic engineering, researchers can even go beyond known existing wild type proteins by introducing useful functional groups thereby tuning or even altering protein sensitivity and selectivity. This can be done by mutagenesis and directed evolution or by incorporation of unnatural amino acids during translation. Additionally, genes for the protein of interest can be inserted in vectors predesigned with handles for affinity purification or immobilization on a surface. Recombinant proteins can then be produced in large amounts in appropriate cellular hosts with very high reproducibility. In addition, chemical reactions involving the amino acids have become standard protocols. Thus, there are thousands of dyes, magnetic beads, nanoparticles or quantum dots designed to form covalent bonds with amino groups, sulfhydryl groups, carboxylic groups and other reactive functionalities in amino acids. It is not uncommon to conduct combinations of techniques, for example, the introduction of fluorescent labels or probes to proteins require in many cases, site-directed mutagenesis followed by covalent bonding of the fluorescent dye. Accordingly, two or more proteins can be combined to create hybrid or fusion proteins with multiple or altered functions. Indeed, research involving the green fluorescent protein and fluorescent proteins of a variety of colors has expanded by leaps and bounds in the last decade. Because these fluorescent proteins can be genetically encoded in cells, it is possible to observe various cellular processes in vivo. However, this topic has been reviewed extensively in the literature and, thus, will not be expounded on in this chapter.