Reporter proteins in whole-cell optical bioreporter detection systems, biosensor integrations, and biosensing applications

Abstract

Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed.

Keywords: aequorin; bacterial luciferase (Lux); bioreporter; biosensor; firefly luciferase (Luc); green fluorescent protein (GFP).

Figure 1.

Bioluminescent reaction catalyzed by the bacterial luciferase gene cassette. A) The luciferase is formed from a heterodimer of the luxA and luxB gene products. The aliphatic aldehyde is supplied and regenerated by the products of the luxC, luxD, and luxE genes. The required oxygen and reduced riboflavin phosphate substrates are scavenged from endogenous metabolic processes, however, the flavin reductase gene (frp) aids in reduced flavin turnover rates in some species. B) The production of light, catalyzed by the products of the luxA and luxB genes, results from the decay of a high energy intermediate (R1 = C₁₃H₂₇).

Figure 2.

Example of a CMOS microluminometer transducer in a hand-held biosensor format. Bioreporter cells engineered to emit bioluminescent light signals are directly interfaced to the transducer element to form a compact and remotely operable biosensor.

Figure 3.

The bioluminescent reaction catalyzed by firefly luciferase. The luciferase protein holds the reduced luciferin to allow for adenylation (a). This process is followed by a deprotonation reaction that leads to the formation of a carbanion (b) and attack by oxygen (c), driving the formation of a cyclic intermediate (d). As this intermediate decays, carbon dioxide is released, forming the excited state luciferin in either the keto (e) or enolate (f) form. Used with permission from Branchini et al. [77].

Figure 4.

The bioluminescent reaction catalyzed by aequorin is dependent on the pre-bound coelenterazine luciferin. Upon calcium binding, the steric orientation of the luciferin is disturbed leading to a cyclization reaction that irreversibly forms a dioxetanone intermediate. As this intermediate decays, carbon dioxide is released and a singlet-excited anion is produced, followed by the generation of light at 465 nm. Used with permission from Jones et al. [94].

Figure 5.

The dual absorption peaks in the GFP spectra are the result of different charge states in the GFP chromophore. The neutral state (left) is responsible for the major peak at 397 nm while the anionic form (right) is responsible for the minor peak at 475 nm. Regardless of the chromophore charge state, emission occurs at 504 nm. Adapted from Scholarpedia.org.

Figure 6.

A) A lab-on-a-CD microfluidic device used in conjunction with GFP bioreporters sensitive towards arsenic. B) A close-up view of the microfluidic channeling that permits sample and bioreporter mixing. Used with permission from Rothert et al. [23].