The possibility of detecting a few molecules using bioluminescence and chemiluminescence is exciting, especially in the context of miniaturized analytical devices.
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A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 3
© 2003 AMERICAN CHEMICAL SOCIETY
Analytical Bioluminescence and Chemiluminescence Aldo Roda Massimo Guardigli Elisa Michelini Mara Mirasoli Patrizia Pasini University of Bologna (Italy)
B
esides the charm of flickering fireflies or the eerie glow of luminescent organisms in seawater, bioluminescence (BL) and chemiluminescence (CL) have an additional special appeal—they are extremely useful analytical tools, with an ever-widening number of novel uses. The analytical peculiarity of BL and CL is that they allow us to “see” an event occurring at a molecular level, that is, a reaction can be observed by watching the photons produced. As little as 30,000 photons/cm2 of retina surface/s are perceivable by human eyes, which means we can see the equivalent of a few thousand reacting molecules. We have instruments able to detect and quantify light emission down to a single photon so that, at least theoretically, the presence of a single molecule can be detected. For imaging analysis, we have ultrasensitive CCD cameras that, in addition to measuring light intensity, can record the pattern of the light emitted from a sample surface with excellent spatial resolution (1, 2). BL and CL are the results of a chemical reaction that leads to electronically excited products that either decay, emitting photons of visible light, or act as sensitizers, passing their energy to another chemical species that will then emit the light. The intensity of the light emitted depends on the overall efficiency of the BL and CL reaction (BL/CL), expressed as BL/CL = CEXF, in which C is the chemical yield, EX is the yield of the excited state, and F is the emission quantum yield of the excited state. BL and CL reactions usually have flash-type kinetics, in which light lasts only few seconds. However, suitable molecules can be added to promote glow-type emission kinetics that are characterized by a steady-state emission lasting several minutes and improve analytical signal handling (modes of triggering and measuring the signal) and measurement reproducibility (3–5). These techniques offer undoubted advantages over more widely used systems, such as fluorescence. The primary advantage is that, because the light signal is generated by a chemical reaction in the dark, a lower nonspecific signal is produced and there is no light scattering. N O V E M B E R 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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FIGURE 1. Phrixothrix hirtus, the railroad worm, with red- and green-emitting luciferases located in its head and lateral lanterns, respectively. (Reproduced with permission from Ref. 6.)
On the other hand, a potential disadvantage of BL and CL systems is that the light-emitting chemical reaction could be uncontrollably inhibited, enhanced, or triggered by sample matrix constituents. In recent years, researchers have extensively investigated BL, including the molecular species involved in light emission, its natural behavior and function in living organisms, and the structure of light-emitting organs. Knowledge of the light organ’s anatomy has facilitated understanding of in vivo signal transmission and the chemically mediated luminescence process. For example, the light-generating organs of many insects consist of rosette-type structures composed of thousands of photocytes that are interpenetrated by nerves and by tracheoles that supply the oxygen. BL is neurally controlled, so it may rely on regulating oxygen entry by the tracheolar end-cells. Living organisms emit light over a wide range of wavelengths, and some even emit two different colors. The railroad worm Phrixothrix hirtus has red- and green-emitting luciferases located in its head lantern and lateral lantern, respectively (Figure 1). In beetle luciferases, color differences are essentially determined by the protein’s primary structure, which in turn affects the activesite microenvironment around the emitter. Most other BL organisms produce different colors through the use of accessory fluorescent proteins or inner filters (6). In fireflies, luciferin and ATP in the presence of oxygen and the BL enzyme luciferase produce light. HO
S
N
N
S
COOH
Luciferase
HO
BL marine bacteria uses a long-chain aldehyde as the luciferin and reduced flavin mononucleotide as the cofactor. On the cutting edge of luminescent technology, cloning the genes that encode BL proteins (firefly, bacterial, Renilla and Gaussia luciferases, aequorin, etc.) has paved the way for developing analytical systems based on ultrasensitive luminescent bioprobes and luminescent reporter genes. Researchers have performed site-directed mutagenesis of the amino acids at the active sites of firefly luciferase, which has helped them understand the BL reaction mechanisms (7 ) and has made available mutated luciferases that display various colors of light. Changes in color emission also can be obtained by modifying the chemical structure of the luciferin substrate. These achievements, along with the ongoing discovery of new BL proteins, herald the development of innovative approaches for molecular recognition, such as simultaneous detection of several analytes or determining various signaling pathways in a cell. Parallel to the recent achievements in BL, highly efficient CL systems based on synthetic CL molecules (luminol and its derivatives, dioxetanes, and acridinium esters) have also been developed. O NH
Peroxidase
NH
H 2O 2, OH–
NH 2 O
+ N 2 + Light – COO NH 2
Luminol
Although the parameters affecting CL efficiency have been improved, the overall quantum yield of CL reactions (CL = 0.001–0.1) remains lower than that of BL systems. However, new acridinium ester analogues, such as N-sulfonylacridinium9-carboxamides, which can be used for direct labeling of biospecific reagents, have recently been synthesized (8), and CL substrates for label enzymes, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), and -galactosidase, have been developed. Because enzyme turnover amplifies a CL signal by 103–105, CL enzyme systems are highly detectable. S
N
N
S
O + AMP + Pyrophosphate + Light
H + ATP O 2, Mg 2+
Luciferin
Luciferase is an oxygenase optimized for light emission that catalyzes the oxidation of luciferin to preferentially obtain oxyluciferin in the singlet-excited state. Luciferase also provides an active-site microenvironment that is favorable to radiative decay of the excited oxyluciferin, thus resulting in very high luminescence quantum yield (BL = 0.8–1.0). Many other luminous creatures use a BL luciferin/luciferase system; the structure of luciferin, the luciferase sequence, and the cofactors vary among different organisms. For example, luciferase from 464 A
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Ultrasensitive labels The sensitivity and detectability of luminescence immunoassays are similar to or better than that of radioisotopes (Table 1; 9). For routine clinical analysis, automated luminescent immunoassays have almost completely replaced radioimmunoassays. Among the various immunoassay formats, CL offers the best performance when applied to sandwich-type assays, the sensitivity of which is determined primarily by the detection limit of the label. The format of most CL immunoassays is heterogeneous, in which
Agents of great shining power Although “cold light” or luminescent phenomena have fascinated humanity for millennia, they were not formally studied until the dawn of the modern scientific method, at a time when alchemists were seeking the philosophers’ stone, which they believed could turn ignoble metals into gold. In 1602, in Bologna, Italy, Vincenzo Casciarolo, a cobbler by trade as well as a dilettante alchemist, discovered and reported on the method for preparing Bolognan phosphorus. His rudimentary process of heating and calcinating yielded its then-mysterious and magical ability to “accumulate light” when exposed to the sun and emit it in the darkness. Casciarolo showed his “lapis solaris”, the sun stone, to many learned men of the time, sparking great interest in this phenomenon throughout Europe. Galileo Galilei was among those who participated in the scientific debate that was stimulated by this discovery. The history of modern marine BL also began at about that time. In 1605, the English philosopher Francis Bacon wrote, “It is not the property of fire alone to give light ... small drops of the [sea]water, struck off by the motion of the oars in rowing, seem sparkling and luminous.” In 1637, René Descartes, a French philosopher and scientist, added, “Striking seawater will generate sparks rather similar to those which are emitted by pieces of flint when they are struck.” The phenomenon of marine BL also fascinated well-known novelists such as Jules Verne. In Twenty Thousand Leagues Under the Sea, he wrote, “[T]he sea seemed to be illuminated all over. It was not a mere phosphoric phenomenon. The monster emerged some fathoms from the water, and then threw out that very intense but mysterious light mentioned in the report of several captains. This magnificent irradiation must have been produced by an agent of great shining power.” Now we know that Bolognan phosphorus is barite (barium sulfate), the “magic” light observed by Casciarolo is inorganic phosphorescence, and seawater luminescence reported by Francis Bacon and René Descartes is BL from marine microorganisms. In the last century many BL and CL phenomena have been thoroughly studied and scientifically explained; nevertheless, the phenomena of luminescence remain a fascinating field of study that still has a magical quality.
the bound and free tracers are usually separated by an immobilized immunoreagent on a solid phase, such as assay tubes, microwell plates, or microparticles. Because the separation step also removes the sample constituents, the subsequent CL reaction will not be affected. Tracers are obtained either by direct labeling of antigens or antibodies with CL molecules or by labeling with enzymes detectable with CL substrates. Alternatively, tracers may be antigens or antibodies labeled with ruthenium(II) tris(bipyridyl), which is detected by electrogenerated CL. Very efficient CL substrates are available for HRP and AP, which are the label enzymes most commonly used in immunoassays. Using dioxetane phosphate substrates, researchers detected 10–20 mol of AP (~6000 molecules), which is the lowest detection limit achieved so far in CL enzyme detection (10). Maeda et al. reported a new BL enzyme immunoassay for hormones that uses acetate kinase as the label and measures ATP, produced by the label-catalyzed reaction, with the conventional luciferin/luciferase system with a detection limit of 1–2 10–20 mol (11). The amount of DNA in a cell is very small: ~5 pg and ~0.5 pg for mammalian and bacterial cells, respectively; both types have millions of kilobase (Kb) pairs. To develop a hybridization assay for a typical 1-Kb gene that uses a 1-Kb probe requires a detection limit of at least 1 ag (1.5 ymol) of nucleic acid. A hybridization assay is basically a sandwich-type assay, so CL can greatly improve its performance. For assays using probes longer than 300 bases, labeling with AP or HRP is recommended, whereas shorter probes can be labeled with haptens that are subsequently immunodetected by enzyme-labeled antihapten antibodies. Commercially available hybrid-capture assays for detecting gene sequences combine hybridization and immunological reactions (12). The essential step of the assay is the capture of the hybrid between an RNA probe and the denatured target DNA sequence by an antibody immobilized on the surface of micro-
well plates that selectively recognizes the RNA/DNA hybrids. The bound hybrids are then detected using a second AP-labeled antibody and a dioxetane phosphate-based substrate. Detection limits are ~1000 DNA copies/mL, which are similar to those obtained with PCR amplification, and there is no risk of crosscontamination. Hybrid-capture assays have been applied successfully to the routine detection of viruses and bacteria as well as gene expression profiles.
Flow-injection and separation techniques Because the analytical performance of CL detection is better than that of other common spectroscopic detection methods, such as spectrophotometry and fluorescence, lower detection limits and wider linear ranges can be achieved. On the other hand, CL detection is not as universal as those techniques and thus can be applied only to a limited number of compounds. However, non-chemiluminogenic compounds can be detected after they have been labeled with CL molecules. CL flow-through detectors are very simple instruments and can easily replace other flow-injection or LC detectors. Unlike spectrophotometers and spectrofluorometers, they do not require light sources, optical filters, or monochromators, because CL emission is very specific to the analyte of interest. In addition, the volumes of flow-through cells for CL detection can be very small, and no particular geometry is required, so spirals or coils of glass or plastic tubing are satisfactory for most applications. However, an optimized flow-cell design can improve the light collection efficiency and signal detectability. Although steady-state CL reactions are optimal for most applications, flash-type CL reactions are usually preferred for flow systems; their rapid kinetics allow collection of as much light as possible in the detector, thus improving sensitivity. Because the reaction is usually triggered when the CL reagents mix with the analyte stream just before it enters the detector, a device to deN O V E M B E R 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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Glass cover Silicon chip CL reagents inlet
gen by the antibody immobilized on the solid phase. On the other hand, the analytical productivity of Sample inlet FIA immunoassays is lower than that of convenSpiral groove for tional immunoassays in the 96- or 384-well microCL measurement plate formats. Nevertheless, improvements leading to a significant increase in assay productivity, such as the development of miniaturized devices, can be envisaged. CL detection has already been applied to micro10 mm fabricated flow-analysis devices to take advantage of Lactate oxidase immobilized Enzyme reactor on controlled-pore glass beads CL’s high detectability and to achieve a considerable reduction in the volumes of both samples and reagents required for analysis. A recently described integrated CL flow cell can measure physiological lactate levels FIGURE 2. Flow-cell assembly and detailed view of the silicon chip in the integrated in a 0.2-µL serum sample in
