Bioluminescent proteins are used in a plethora of analytical methods, from ultrasensitive assay development to the in vivo imaging of cellular processes. This article reviews the most pertinent current bioluminescent-protein-based technologies and suggests the future direction of this vein of research. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.)Since the dawn of civilization, humans have shown enormous interest in exploring the world surrounding us. Our curious nature, fueled by a desire to both understand and control natural phenomena, has spurred the development of techniques and tools that constitute the foundation of today's analytical chemistry. In the past 100 years, analytical science has progressed from crude techniques such as filtration and distillation to highly sophisticated techniques such as atomic force spectroscopy, surface plasmon resonance, and chemometrics-based signal deconvolution algorithms. With these developments, our ability to observe and analyze has progressed from the macroscopic world visible to the naked eye to the microscopic domain, which must be magnified with optical lenses, and now to the nanometer scale and beyondsa feat that has piqued the interest of engineers, physicists, biologists, and chemists alike. Observing and quantifying events at these miniscule dimensions present new challenges and require a diverse array of specialized tools. In that regard, light-emitting proteins are invaluable for detection and imaging, as well as for revealing the properties of these nanoscale environments.Light has inspired many cultural superstitions: early Polynesians, Scandinavians, and ancient seafarers all wove tales and mythologies about the lights and fires they beheld over water and in fields and mountains. Because they were unable to rationally explain these illuminations, they attributed the mysterious lights to machinations of the gods. Many notable philosophers and explorers, from Aristotle to Christopher Columbus, also observed "cold lights"swhat we know now as bioluminescence. As logic took hold in the age of reason, scientists such as Robert Boyle and Charles Darwin attempted to rationalize the existence and purpose of bioluminescent phenomena. Today, we recognize bioluminescence as the production and emission of light by a living organism. An internal reaction converts chemical energy into lightsa reaction almost always catalyzed by a protein.A large variety of bioluminescent proteins of many biological origins and evolutionary functions have been studied, and their reaction mechanisms, substrates, and bioluminescent properties vary widely. Researchers group bioluminescent proteins into two major categories: photoproteins and luciferases (Figure 1). Photoproteins are bioluminescent proteins that are capable of emitting light in proportion to the concentration of protein, whereas in luciferase-catalyzed reactions, the amount of light emitted is proportional to the concentration...

786 1. Introduction 786 2. Organization of the Ideas Presented and Discussed at the Conference 787 2.1. Definition of key terms 787 3. Martian Environments Deemed to Be Most Prospective for Extant Martian Life 788 3.1. Caves 790 3.2. Deep subsurface 790 3.3. Ice 793 3.4. Salts 796 4. Methodologies: A Summary of Possible Approaches and Strategies to Search for Extant Life on Mars 797 4.1. Geologically guided search strategies 797 4.2. Possible detection methods for extant martian life 799 4.3. Possible constraints relevant to the search for extant martian life derived from laboratory experiments 802 5. Discussion 804 Acknowledgments 805 References 805

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Environmental pollution is both a worldwide and a local issue, and microplastic pollution in particular is receiving increased attention due to its prevalence and bioaccumulation potential affecting the food chain. This laboratory experiment uses current, research-based methods such that the students can determine the extent of microplastic pollution in local soil samples. This laboratory experiment can be used as either a 2 or 3 week mini-research-project for first-year undergraduate students in either an introductory chemistry course for nonmajors or a general chemistry course for majors. The laboratory experiment gives students exposure to sieving, density gradients, and exposure to the Fenton reagent to isolate microplastics from soil samples, which are then analyzed and quantified under stereomicroscope magnification. Several general chemistry topics common to most first-year chemistry courses (density and solution concentration calculations, etc.) are emphasized during the laboratory experiment. From postexperiment assessments, students showed a marked improvement in select skill sets and knowledge of the microplastic pollution problem, and some students recognized their misconceptions concerning research following the completion of this laboratory experiment.