Detector Sensitivity

An important characteristic of a visible light detector is its wavelength-dependent response. There are many different ways that vendors report the wavelength-dependent response of the detectors they offer. Direct comparison of detectors can be done only after the methodologies have been examined carefully. Even if a published response curve is taken to be truly representative of a certain aspect of the detector's performance, it may not directly indicate the relationship between input and...

Location of the Interaction Between Y941 and SH2n

Spontaneous emission of luminescence by a particular PPI-induced complementation of the split Renilla luciferase together with its membrane-permeable substrate, coelenterazine, allows noninvasive imaging of the sites and time of its occurrence in living cells. The PPI between Y941 and SH2n in the CHO-HIR cells expressing sRL91 was thereby imaged with and without insulin stimulation as shown in Fig. 4.5a. Upon 100-nM insulin stimulation, luminescence emitted by complement Renilla luciferase...

Selecting the Right Detector

Table 11.3 presents some broad generalizations about the differences between vacuum-based and solid-state detectors. Solid-state devices have earned the reputation of being superior to vacuum-based devices, but there are notable exceptions to this situation. Vacuum-based detectors usually have a much lower cost per unit of active area than comparable solid-state detectors. In addition, certain types of photocathodes can achieve a lower noise level per unit area than even the best-cooled...

Resonance Energy Introduction

In the ocean, bioluminescence is found across nearly all taxa, from bacteria to fish (Fig. 2.1 and review by Herring 1987). In many senses, it is easier to name the few non-luminous phyla than it is to detail each luminous group. However, even in light of recent breakthroughs in molecular biology, we are hardly closer than E. Newton Harvey (1952) was to being able to explain why luminescence is distributed among phyla the way it is. For example, among the phytoplankton, many dinoflagellates are...

BRET Principle Efficiency and Instrumentation

The efficiency of BRET is dependent upon factors such as spectral properties, relative distance, and orientation of donor and acceptor molecules 6, 7 . BRET normally occurs when the distance between the donor and the acceptor is within 100 A, and the efficiency of BRET is inversely related to the distance to the sixth power. The rate of energy transfer is given by F rster's equation, where fcj. is the rate of energy transfer, Td is the fluorescence lifetime of the donor in the absence of the...

LuxCDABE Bioreporters

These bioreporters contain the full lux operon cassette, thereby allowing for a completely independent bioluminescent system that requires no external addition of a substrate. In this biosensing system, the bioreporter is exposed to the target analyte and a quantitative change in bioluminescence is detected, often within an hour. The rapidity of the assay, its ease of use, and the substrate-free characteristic of this system make luxCDABE bioreporters ideal for real-time, online, field, and...

Biotin Avidin Binding Assays

Biotin (vitamin H) is a vitamin found in tissue and blood that binds to the glycoprotein avidin, which contains four identical binding sites for biotin. Biotin and its binding protein avidin have been used extensively in the development of a variety of bioanalytical techniques that take advantage of the extremely high affinity (ka 1015 M-1) between the two biomolecules. Biotin has also been selected as a model analyte to develop binding assays in order to explore the possibility of applying...

Transmission Efficiency

Transmission efficiency indicates how much light is lost because of reflections at optical surfaces and absorption within the lens elements. Most high-quality commercial lenses use antireflection coatings on the surfaces to minimize reflective losses. Most lens materials also transmit well in the 390-700 nm visible light spectrum. However, because the transmission efficiency of the total optical system is the product of the independent transmission efficiencies of each optical element, it is...

Methods of Coupling the Signal to the Detector

An important aspect of experimental design is the optical system used to collect light from the sample and direct it to the detector. The four general categories for optical systems shown in Fig. 11.2 will be considered (1) proximity focused, (2) microscope objective, (3) macro lens, and (4) fiber optic. In some cases the physical constraints of the sample force selection of a certain configuration. Key aspects of each configuration will be introduced, and a discussion of how to evaluate the...

FRET and BRET Techniques

Most of these assays involve resonance energy transfer (RET) techniques. RET is a short-range, nonradiative energy transfer between donor and acceptor molecules that takes place only if the two species are in close proximity (< 10 nm) to each other. RET systems are therefore spectroscopic rulers that are suitable for monitoring interactions between two partners, provided that each component of the donor-acceptor couple is linked to one partner. The same strategy can be used to detect protein...

Luminescent Proteins in Binding Assays 155

Aldo Roda, Massimo Guardigli, Elisa Michelini, Mara Mirasoli, and Patrizia Pasini 9.2 Protein-Protein and Protein-Ligand Interaction Assays 158 9.2.1 FRET and BRET Techniques 158 9.2.2 FRET and BRET Applications 159 9.2.3 Other Detection Principles 163 9.3 Antibody-based Binding Assays 163 9.3.4 Expression Immunoassays 166 9.3.5 BRET-based Immunoassays 167 9.4 Biotin-Avidin Binding Assays 168 9.5 Nucleic Acid Hybridization Assays 169 9.7 Concluding Remarks 172 References 173

Discovery of Photoprotein

In 1961, we found an unusual bioluminescent protein in the jellyfish Aequorea and named it aequorin after its genus name (Shimomura et al. 1962). The protein had the ability to emit light in aqueous solutions merely by the addition of a trace of Ca2+. Surprisingly, it luminesced even in the absence of oxygen. After some studies, we discovered that the light is emitted by an intramolecular reaction that takes place inside the protein molecule, and that the total light emitted is proportional to...

Low Light Levels

Visible light is a form of energy that can be observed by the human eye. It has been observed that visible light energy occurs in discrete units, and these units are called photons. Photons can be theoretically treated as particles having an energy E proportional to their wavelength X as follows where h is Planck's constant and c is the speed of light in a vacuum. The human eye perceives light of different wavelengths as different colors as shown in Table 11.1, which is provided as an...

Ackowledgments

This work was funded by the National Institute of Health (NIH), the National Science Foundation (NSF), the National Aeronautics and Space Administration (NASA), and the National Institute of Environmental Health Sciences (NIEHS). 1 Ismagilov, R. F. Angew. Chem. Int. Ed. 2 Meldrum, D. R., Holl, M. R. Science 2002, 297, 1197. 3 Erickson, D., Li, D. Anal. Chim. Acta 4 Hansen, C., Quake, S. R. Current Opinion in Structural Biology 2003, 13, 538. 5 De Mello, A. J. Anal. Bioanal. Chem. 2002, 372, 12....

Probes Used for in Vivo Bioluminescence Imaging

In vivo bioluminescence imaging (BLI) is a powerful tool that can be used to study biological events temporally and noninvasively in live animals. Biological entities such as bacteria, tumors, or other cells or genes are labeled with bioluminescent reporters and light is detected externally using a highly sensitive cooled charged-couple device (CCD) camera 7 . As a bioluminescent probe, used in in vitro and in vivo assays, luciferase can generate visible light by oxidation of an enzyme-specific...

Other Luciferins Known and Unknown

Although the four types of luciferin listed above can account for a large portion of the phylogenetic diversity of marine bioluminescence, there are several other taxa whose light-producing chemistries are either unique or as yet unknown. Some of these organisms are described below. The bivalve Pholas, a rock-boring clam, was one of the first organisms to have its luminescence chemistry investigated in detail (Dubois 1887). Its chemistry is a variation on the ones presented thus far the...

Coelenterate Photoproteins

Several kinds of photoprotein, including aequorin and obelin, were isolated from hydrozoan jellyfishes and hydroids. All of them emit blue light when Ca + is added, regardless of the presence or absence of oxygen. The coelenterate photoproteins are suitable for use in the detection and measurement of trace amounts of Ca2+, and aequorin has been widely used in the studies of Ca + in various biological systems, including single cells (Blinks et al. 1976 Ashley and Campbell 1979). The overwhelming...

Ophiopsila Photoprotein

The brittle star Ophiopsila californica is abundant around Catalina Island, off the coast of Los Angeles (Shimomura 1986a). An animal of average size weighs about 3-4 g, and has five arms of about 10 cm long. The purified photoprotein luminesces in the presence of H2O2, emitting a greenish-blue light (Amax 482 nm). Molecular oxygen is probably not needed for the luminescence reaction. The molecular weight of Ophiopsila photoprotein is estimated to be about 45 000 by gel filtration. The...

Miniaturization and Microfluidics

The need for fast, accurate, and low-cost analytical methods for the high-throughput analysis of complex and low-volume samples is fueling up advancements in the area of microfluidic devices. Great efforts have been made toward the miniaturization of conventional bench-top analytical techniques into microfluidic chip-based formats 1 . These microfluidic chips are based on architectures of microreservoirs and microchannels (Fig. 10.1) with very small dimensions, which range from tens to hundreds...

Biophotonic Imaging in Animals A Living Light on Diseases

One of the most exciting and promising applications of beetle luciferase genes is the real-time imaging of normal and pathogenic biological processes in living organisms (Fig. 3.5). a. Transgene b. Bacterial c. Gene d. Lymphocyte Expression Infection Transfer Trafficking Fig. 3.5 Biophotonic imaging of bioluminescent mice according to Contag and Bachmann 61 . Fig. 3.5 Biophotonic imaging of bioluminescent mice according to Contag and Bachmann 61 .

Protein Splicingbased Split Firefly Luciferase System [23

Here we describe a protein splicing-based split luciferase to monitor proteinprotein interactions in mammalian cells, using an intein of DnaE derived from the cyanobacterium Synechocystis sp. strain PCC6803. The DnaE intein was selected among several known inteins because it has the natural splicing ability to ligate accompanying proteins in trans. This principle is shown in Fig. 4.1. The Ssp DnaE intein is a naturally split intein, composed of 123 amino acid residues in the N-terminal half and...

Startle or Distract

There are two mechanisms that have been offered to explain the function of dinoflagellate bioluminescence (and both are similarly applicable to other organisms). The first is that the sudden flash of light startles a predator or triggers a rapid rejection response before the cell is ingested (Buskey et al. 1983, 1987). This kind of flash could also apply to jellies, which may be trying to minimize the damage caused by an encounter, or advertising their toxicity. Disruptive flashes are seen in...

Semisynthetic Aequorins

The core cavity of the aequorin molecule can accommodate various synthetic analogues of coelenterazine in place of coelenterazine. The coelenterazine moiety in native aequorin can be replaced by a simple process. First, aequorin is luminesced by the addition of Ca +, and then the apoaequorin produced is regenerated with an analogue of coelenterazine in the presence of EDTA, 2-mercaptoethanol (or DTT), and molecular oxygen. The products are called semisynthetic aequorins and are identified with...

Prey Attraction

Probably the most intuitive role for bioluminescence is also the rarest. This is the use of bioluminescence to lure prey. Such a function has been suggested for anglerfish, with their luminous organ, and for some squids. Unfortunately, witnessing a capture resulting from a glowing lure is nearly impossible in the deep sea. Recently, another example of glowing lures was discovered in a deep-sea, fish-eating siphonophore (Haddock et al. 2005). Clearly, bioluminescence is an important component of...

Sea Pansy Luciferase

The sea pansy Renilla reniformis is a coelenterate that, unlike other bioluminescent organisms, emits a bright green light. In vivo, Renilla luciferase oxidizes its substrate coelenterazine, and after energy transfer to the green fluorescent protein, light is emitted at 509 nm. In vitro, it is represented by the Rluc gene and produces a blue light of 482 nm 27 . Like other luciferases, Renilla luciferase has no endogenous activity in bacteria and thus can find applications in bacteria-based...

Dinoflagellate Luciferin

A sailor or beachgoer who sees sparkling lights in the water at night is most likely witnessing the bioluminescence of dinoflagellates. These protists occupy a somewhat equivocal position between unicellular algae and protozoans. Some species are autotrophic, deriving energy from photosynthesis others are heterotrophic, consuming diatoms and other plankton, and some have qualities of both. Some types are even parasitic and may cause severe health hazards, but these are not known to be...

Bacterial Luminescence

Luminous bacteria (Fig. 2.3A) such as Vibrio fischeri produce a low, steady glow for hours in the presence of oxygen. In the best-studied species, transcription of light-producing genes is regulated by the presence of an autoinducer in the environment (Nealson and Hastings 1979). This autoinducer is produced by the bacteria and accumulates as the population density increases, so that a dilute culture will not produce appreciable light. The genetic cassette responsible for light production is...

Inhibitors of Aequorin Luminescence

All thiol-modification reagents cause weak, spontaneous luminescence of aequorin in the absence of Ca +, as already mentioned. They are in effect inhibitors of the Ca +-triggered luminescence of aequorin, because the quantum yields ofaequorin in the luminescence caused by these reagents ( 0.008) are much lower than that of the Ca +-triggered luminescence of aequorin (Shimomura et al. 1974). Bisulfite, dithionite, and p-dimethyaminobenzaldehyde are all strongly inhibitory even at micromolar...

Examples of BRET Donor Acceptor Pairs

Renilla luciferase (RLuc) was originally isolated from the anthozoan coelenterate Renilla reniformis 4, 14 . It catalyzes the oxidative decarboxylation of the substrate coelenterazine to produce coelenteramide and light (wavelength maxima 480 nm) (Fig. 6.3). Renilla luciferase has been used as a reporter gene for studying in vitro and in vivo gene regulation. Native coelenterazine is the natural substrate for Rluc however, it can also utilize analogues of coelenterazine as substrates yielding...

GFP in Miniaturized Microfluidicbased Assays

Because of the inherent advantages of GFP, this fluorescent protein has been successfully employed in numerous small-volume assays, including microassays integrated onto microfluidic platforms. One such example includes the development of a microfluidic assay employing the GFP mutant GFPuv for the detection of arsenite and antimonite 74 . In this work, a whole-cell assay was developed incorporating the gene for the reporter protein GFPuv. For that, a plasmid that encoded the gene for GFPuv was...

Magnification

Optical systems with higher magnification tend to have higher numerical apertures, while optical systems with greater reduction (i.e., increased field of view) tend to have lower numerical apertures. However, choosing a higher magnification optic to achieve a higher numerical aperture is not always a sound approach. Even though more light is collected, it is spread out over a larger area, affecting the apparent brightness of the image Consider the difference between a 5x 0.25 NA lens and a 20x...

References

F., Rivera, H., Gray, J., McCann, R. O., O'Kane, D., Cummings, R. D., Cormier, M. J., Smith, D. F. Use of recombinant biotinylated aequorin in microtiter and membrane-based assays purification of recombinant aequorin from Escherichia coli. Biochemistry 1992, 31, 1433-1442. 2 Galvan, B., Christopoulos, T. K. Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA. Anal. Chem. 1996, 68, 3545-3550....

Luciferase

Luciferase is a bioluminescent protein that exists in diverse organisms such as bacteria, insects, and marine coelenterates. Because these systems have evolved independently, homology is not typically observed among the luciferase genes from different groups. Therefore, the most commonly used luciferase genes are distinguishable based on the bioluminescent organism from which they have been isolated, using the abbreviations lux (bacterial), luc (firefly), and Rluc (sea pansy Renilla) 100 . As a...

Mechanism of Aequorin Luminescence and Regeneration of Aequorin 1451 Structure of Aequorin

Aequorin is a globular protein with three EF-hand domains to bind Ca +, and it accommodates a peroxidized coelenterazine in the central cavity of the protein (Head et al. 2000). The presence of a peroxy group bound to position 2 of the coelenterazine moiety was previously suggested (Shimomura and Johnson 1978) and confirmed by 13C nuclear magnetic resonance spectroscopy (Musicki et al. 1986). The protein conformation of aequorin is much more compact and rigid than that of apoaequorin,...

Cypridina Vargula Luciferin

Ostracods (Fig. 2.5D) are small crustaceans enclosed in a clam-like chitinous carapace. The luciferin of Cypridina (also called Vargula) was one of the first to be crystallized and chemically characterized (Kishi et al. 1966 Shimomura et al. 1957). Also known as seed shrimp or firefleas, ostracods are the source of one of the most famous anecdotes about bioluminescence. During World War II, the Japanese army collected massive quantities of ostracods and dried them on tarps on the shore. These...

Recombinant Aequorin

The cloning and expression of apoaequorin cDNA was accomplished by two independent groups in 1985. One of these groups analyzed the cDNA clone AQ440 they obtained and reported that apoaequorin is composed of 189 amino acid residues (Mr 21 400) with an NH2-terminal valine and a COOH-terminus proline (Inouye et al. 1985, 1986), which is consistent with the results of the amino acid sequence analysis of native aequorin reported by Charbonneau et al. (1985). In contrast, the other group reported...

LuxAB Bioreporters

These bioreporters contain only the luxA and luxB genes, which are sufficient for generating the bioluminescent signal however, because the genes that code for the production of the substrate are not present, the substrate must be supplied to the cell. Typically, decanal is added at some point during the assay. This system has the advantage that one can control the moment when the reaction is triggered and the measurement be taken right after, dramatically reducing the basal bioluminescence...

Coelenterazine

Coelenterate-type luciferin is by far the most widely distributed marine luciferin, found in at least seven different phyla, from protists to vertebrates. Like the Cypridina luciferin discussed above, coelenterazine is a nitrogen-based imidazole compound. While it has been detected in numerous species (Shimomura et al. 1980 Thomson et al. 1997), its presence does not necessarily imply that it is responsible for light production, as it has been found in many non-luminous species as well...

Comparison of BRET and FRET

Fluorescence resonance energy transfer (FRET), in which both the donor and acceptor molecules are fluorescent proteins or chemical probes, is a very popular biochemical technique employed in a variety of applications 8 . Several FRET pairs have been well studied and allow for a range of selection in terms of fluorescence excitation-emission wavelengths. In FRET studies, there is no need for addition of substrate to generate a signal. High signal levels can be achieved by selecting suitable...

FRET and BRET Applications

The great variety of available fluorescent and bioluminescent proteins emitting at different colors and the possibility to produce genetic in-frame fusions have had a great impact on FRET applications (which were originally based on organic fluorophores) and made BRET possible. Further advancements came as a result of the availability of improved substrates for bioluminescent proteins. For example, Packard has developed a BRET technology (BRET2) based on a proprietary coelenterazine substrate...

Luminous Taxa

Although there are numerous examples of the independent evolution of bioluminescence, four types of luciferin are responsible for light production in the majority of marine phyla (Fig. 2.2). These light-emitting compounds are conserved, while the catalyzing proteins (either luciferases or photoproteins) are diverse and encoded in the genome of each luminous organism. One explanation, perhaps the most parsimonious, for the few types of luciferins in many phyla is that they are passed along...

Numerical Aperture

The light collection efficiency of an optical system is largely determined by a quantity known as numerical aperture. The numerical aperture of an optic is described as where n is the index of refraction of the media between the sample and the optic (nair 1.0, nwater 1.3, noil 1.51) and a is the angular aperture of the system, measured as the half-angle included by a cone with its apex at the sample and its base at the perimeter of the first surface of the collection optic when the sample is in...

Protease Sensors

Proteases are key enzymes that are implicated in a number of cardiovascular, oncological, neurodegenerative, and inflammatory diseases. For instance, in cancer, proteases are thought to be involved in the invasion, metastasis, and angiogenesis of tumor cells 75 . Cancer treatments are being sought that target these proteases and inhibit their activity. For example, matrix metalloproteases (MMP) and cathepsins have been found to enable malignant cells to cross the basement membrane and digest...

Aequorin

Emission Xmax 469 nm Detection limit 1 x 10-18 M Senseomics www.senseomics.com MP Biomedical www.mpbio.com Invitrogen www.invitrogen.com LUX Biotechnology www.luxbiotech.com Chemicon www.chemicon.com ChemFLASH Streptavidin Conjugate Pack Aequorin conjugated with streptavidin DemoLite Plate (flash luminescence test plate) Serial dilutions of lyophilized aequorin Demonstration of instrument linearity Universal Amplicon Detection AssayChem FLASH AquaLite Contains anti-FITC monoclonal antibody...

Novel Luciferases Different Colors for Different Occasions

Until recently, only firefly luciferases had been used for bioanalytical purposes. However, they produce only yellow-green light under physiological conditions and have the drawback of being pH sensitive, reducing the efficiency of signal detection for most applications. Red luciferase mutants were produced by genetic engineering, and some of them are currently in use for applicative purposes. However, their spectrum is not very red-shifted and furthermore is very broad 36, 85-87 . Click beetle...

Split Luciferase Works as a Probe for Protein Interaction

To examine whether some particular PPIs in eukaryotic cells facilitate the splicing event to yield matured luciferase, phosphorylation of IRS-1 and its target SH2N domain derived from phosphatidyl inositol 3-kinase, both of which are involved in physiologically relevant insulin signaling, was chosen as a known protein interaction pair 25 . After the CHO-HIR cells were transiently co-transfected with pIRES-DSL(Y S) and pRL-TK vectors, the cells were stimulated with 1.0 X 10-7 M insulin for 72 h,...

Nucleic Acid Hybridization Assays

The analysis of specific nucleic acid sequences by hybridization is a fast-growing area of laboratory medicine. These methods are based on the specific hybridization of a suitably labeled oligonucleotide probe with a target nucleic acid sequence, followed by the hybrid detection and quantification. Luminescent proteins are becoming more and more popular as labels because luminescence-based hybridization assays are amenable to automation and offer higher detectability over conventional...

Dual and Multiple Reporter Assays

For practical purposes, two promoter activities, for target and control genes, must be analyzed, because expression of the reporter enzyme depends on conditions in Fig. 3.4 Multicolor reporter system showing the simultaneous monitoring of mRORa4 dose-dependent induction of RORE-mediated (red bars) and mBmall promoter fragment-driven (orange bars) transcriptions 60 . Fig. 3.4 Multicolor reporter system showing the simultaneous monitoring of mRORa4 dose-dependent induction of RORE-mediated (red...

Luminescence Reaction

In the case of aequorin reacting with Ca +, a conformational change of protein takes place when one molecule of aequorin is bound with two Ca2+ ions (Shimomura 1995b). The conformational change results in the cyclization of the peroxide of coelenterazine into the corresponding dioxetanone, which instantly decomposes and produces the excited state of coelenteramide and CO2 (Shimomura et al. 1974 Shimomura and Johnson 1978). When the energy level of the excited state of coelenteramide falls to...

Green Fluorescent Protein

Fluorescent Protein Structure

The green fluorescent protein (GFP) is a naturally occurring fluorescent protein that is isolated from the jellyfish Aequorea victoria and some other marine organisms 68 . The role of GFP in nature is to shift the blue bioluminescence emitted by aequorin into the green region. Such a shift improves the quantum efficiency and penetration of light through water and reduces light scattering. GFP from Aequorea victoria is a 238-amino-acid protein, with a molecular weight of approximately 27 000 Da....

Extraction and Purification of Aequorin

Aequorin is the best-known photoprotein and has been used widely in various applications. The first step in the extraction of aequorin from the jellyfish Aequorea (average body weight 50 g) is to cut off the circumferential margin of umbrella that contains light organs, making about 2-mm-wide strips commonly called rings. This process is important because it eliminates about 99 of unnecessary body parts that do not contain aequorin. The rings can be made efficiently by using specially made...

Polynoidin

A membrane photoprotein isolated from the scales of the scale worm Harmothoe lunulata was named polynoidin Nicolas et al. 1982 . The purified photoprotein Mr 500 000 emits light in the presence of molecular oxygen Amax 510 nm by the action ofsodium hydrosulfite, the xanthine-xanthine oxidase system, Fenton's reagent H2O2 plus Fe , or other reagents that produce superoxide radicals. The photoprotein luminescence was 30 brighter in phosphate buffer than in Tris buffer, and the luminescence...

Luminodesmus Photoprotein

This is presently the only example of a photoprotein of terrestrial origin. The millipede Luminodesmus sequoia Loomis and Davenport 1951 emits light from the surface of its whole body continuously day and night. The photoprotein extracted and purified from this organism emits light Amax 496 nm when ATP and Mg are added in the presence of molecular oxygen Hastings and Davenport 1957 Shimomura 1981 . Thus, the luminescence system of Luminodesmus resembles that of the fireflies in that it requires...