Chemiluminescence in Analytical Chemistry

Figure 16 Schematic layout of microchip designs for (a) PCRD1 and (b) PCRD2; dimensions in mm. Letters are referred to in the text and identify the solution introduction reservoirs and points where potentials were applied. Indicated channels dimensions are for 10-m-deep devices, and are not repeated in (b) except where differing from those in

(a). (From Ref. 107, with permission.)

voirs C and E contain buffer with added luminol. A potential of 5 kV is applied to E, with A and B at ground and D at 1.1 kV. This produces a continuous stream of CL product flowing past the detector. The chip is translated in the x and y directions and the microscope in the z direction to maximize signal. The optimum occurs with the lens centered 0.1–1.0 mm from the Y intersection. The double-T injection is performed for 30 s, with 1.2 kV applied between A (at

ground) and B, forming a geometrically defined sample plug of about 60 pL. Much larger plugs are formed with a single-T injection, which uses the same first step as the double-T, followed by 0.5–5 s with 3 to 6 kV at E, 1.2 kV at C, and A and B at ground. In both cases, separation uses 6 kV between B and E, with D at 1.3 kV. The detection limit obtained, 7–35 nM for HRP F1 for onchip CL, is about 50to 100 fold lower than could be achieved for absorbance detection [109]. Using this microchip, separation and CL detection of the products of an immunological reaction of a fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG were performed.

4.2 Compact Detection Cells

Although research on the coupling of CE with CL detection has increased in recent years, the technique remains problematic. Most of the CE-CL detectors reported have involved variations of a postcapillary reactor to mix the reagents. The reactor requires insertion of the separation capillary into the reaction/detection capillary. These procedures are manually intensive and it is difficult to reproducibly control reagent concentrations at the reactor. To overcome these problems, a novel compact CL detection cell, made of PTFE, has been recently designed for CE [110]. This detection cell is equipped with an optical fiber, a fused-silica capillary, and a grounding electrode and it could easily be combined with CE equipment without any complex construction, expensive implements, tedious procedures, or special techniques (Fig. 17). The CL light generated at

Figure 17 Schematic diagram of the CL detection cell. (From Ref. 110, with permission.)

the capillary outlet was transported by the optical fiber to a PMT. The luminol CL system was adapted for the use of this cell, carrying out detailed optimization of the concentration of hydrogen peroxide and catalyst. Using the cell, authors have obtained a detection limit for luminol of 5.10 10 M, which is the most sensitive result reported to date. Also a mixture of glycine, glycylglycine, and glycylglycylglycine, which was labeled with isoluminol isothiocyanate (ILITC), was sensitively detected and baseline-separated. Recently, the same group has developed a simpler, more convenient batch-type cell without an optical fiber for CE, to which only a fused-silica capillary and a grounding electrode were inserted [111]. In this sense, the cell also works as an outlet reservoir including the migration buffer and it is placed just in front of a photosensor module that captures directly the light generated at the capillary. Luminol, ILITC-labeled compounds, H2O2, and the catalyst are dissolved in a 10-mM phosphate buffer that is used as electrophoretic buffer. The catalyst is added to the inlet reservoir and H2O2 to the outlet reservoir (detection cell). Sample injections are performed by gravity for 10 s at a height of 20 cm. The sample migrates in the migration solution toward the CL detection cell and mixes with reagents generating the CL emission, which is captured by the detector. These CE-CL systems are expected to become a practical CL detection system for CE.

4.3 Online Solid-Phase CL Detector in CE

Recently, a novel online solid-phase CL detector has been designed for CE based on the strong CL signal observed when adding trace amounts of luminol, ABEI, or lucigenin in solution to milligram amounts of BaO2 solid powder [112]. The oxidation reactions of luminol, ABEI, or lucigenin are relatively fast and the CL mechanism is considered to involve reactive oxygen species (O•2 ) existing on the surface of BaO2 particles. The environment of the BaO2 particles influenced the CL intensity significantly. Separations were carried out in 0.075 mm id 0.375 mm od fused silica capillaries with a total length of 27 cm. The detection section was 0.3 cm in length. The preparation of this online CE-CL was finished in two steps, packing the BaO2 powder into the detection window section and filling the electrolyte solution into the capillary using a microsyringe. Sample introduction was achieved by electroinjection method at a constant voltage (200 V/cm) for a fixed period (5–10 s). The light generated was collected onto a PMT connected to the recorder to produce the electropherogram. To avoid the BaO2 particles moving from the detection window, the flow rate of the buffer solution must be controlled at lower than 1 cm/mm. Using a pH 8.3 0.05 M sodium borate solution as running buffer with 5% of acetonitrile, luminol, lucigenin, and ABEI were detected online after separation, providing detection limits of 1 10 8 M, 5 10 8 M, and 7 10 8 M, respectively.

5. CONCLUSIONS

The versatility and the robustness of CE separation in conjunction with the extreme sensitivity inherent to CL-based reactions make a combination of both techniques promising for application in a wide range of fields, including environmental analysis, biomedicine, and biological research and practice. Obviously, in comparison with other detection modes widely incorporated in CE, CL detection is a slowly evolving technique and advances should focus on the development of new detectors that are instrumentally simpler than existing systems and that offer the ability to detect various types of analytes at trace levels.

With the advancing automatization and computerization of CE instruments, the application of micromachining techniques, and the improvement of the devices for coupling CE with CL detection, it is hoped that both techniques may be incorporated in the future as suitable methodology in routine laboratories, being complementary to classical techniques such as HPLC and offering new alternatives to the analytical chemist.

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Bioluminescence (BL) is a type of CL that occurs naturally in living organisms and is also used in vitro. Measurement of light from a chemical reaction is very useful from an analytical point of view, because in appropriate experimental conditions the light output intensity is directly related to the analyte concentration, thus allowing precise and accurate quantitative analysis. In addition, the kinetics of light emission is usually a steady-state glow type, which simplifies sample handling and measurement procedures.

CL as an analytical tool has several advantages over other analytical techniques that involve light (mainly absorption spectroscopy and fluorometry): high detectability, high selectivity, wide dynamic range, and relatively inexpensive instrumentation.

The superior detectability of CL and BL measurements is partly due to a low background. In luminescence measurements two components of light reach the detector: the first one (i.e., the net analytical signal) is proportional to the analyte concentration, while the second component (i.e., the background) is an approximately constant light level due to various factors such as the phosphorescence of plastics, impurities in the reagents, emission from other sample components, detector dark current. Warmup and drift of light source and detector and interference from light scattering present in absorption and fluorescence methods are absent in CL, making the background light component much lower and thus achieving a significant gain in sensitivity.

Selectivity derives from the fact that the analyte of interest generates its signal in the presence of compounds that normally interfere in fluorescence measurement and that do not themselves produce light when the chemiluminescent reagents are mixed together.

Wide dynamic ranges allow samples to be measured across decades of concentrations without dilution or modification of the sample cell. This is due to the way the chemiluminescent signal is generated and measured, i.e., using no excitation source for light production and a phototransducer with an inherent wide range of response for light detection. The light emitted from chemiand bioluminescent reactions is typically measured using a luminometer. Luminometers are simple, relatively inexpensive instruments designed to measure sample light output, generally by integrating light emission for a given period. All luminometers basically consist of a sample chamber, a detector, and a signalprocessing apparatus, and are used to measure emission from different sample formats (the most common being single tubes and microtiter plates).

Photodiodes and photomultiplier tubes (PMTs) are the detection devices commonly found in commercial luminometers. Even if improvements in photodiodes have made them suitable for some applications, PMTs are still the detectors of choice for measuring extremely low levels of light.

A relatively recent advancement in light-detection technology for analytical purposes is represented by low-light imaging devices based on intensified Vid-

icon tubes or high-sensitivity charge-coupled devices (CCDs). These luminescence imaging instruments, also known as luminographs, allow not only the measurement of light intensity at the single-photon level, but also the spatial distribution of the light emission on a target surface to be evaluated.

CL is utilized in various analytical techniques in which small amounts of analytes are detected and quantitated by measurement of the light emission [1– 3]. Chemiluminescent reaction systems often involve enzymes, such as alkaline phosphatase (AP) and horseradish peroxidase (HRP), and suitable CL substrates that allow for the detection of enzymes with very high efficiency [4–7]. These enzymes are widely used as labels in the development of immunoassays [8, 9], blotting [10, 11], and gene probe assays [12, 13]. Coupled enzymatic bioand chemiluminescent analytical methods have also been developed: in particular, ATP-involving reactions (kinases) have been coupled with the firefly luciferinluciferase system, NAD(P)H-producing or -consuming enzymatic reactions (dehydrogenases) with bacterial luciferases, and the luminol/H2O2/HRP system has been coupled with oxidase enzymes [14–18]. Advances in molecular biology and the increasing need for ultrasensitive assays have led to the development of novel luminescence systems for a wide variety of applications in genetic research, food technology, environmental monitoring, and clinical chemistry, so that CL-based immunoassay and gene detection kits became commercially available and routinely used [8, 9, 12, 13].

CL imaging also represents a promising detection system that is increasingly used for ultrasensitive quantitation and localization of analytes in a wide range of applications [19, 20]. CL imaging is suitable for filter membrane biospecific reactions, such as the southern, northern, or western blot tests, and dot blot hybridization reactions. In these techniques nucleic acids or proteins are either blotted on filter membrane after separation by gel electrophoresis, or directly dotted on the membrane. Nucleic acids are then hybridized with a complementary gene probe labeled with a hapten and detected by antihapten antibody conjugated with an enzyme and CL substrate; proteins are detected by specific antibody followed by antiantibody conjugated with an enzyme and CL substrate. The main advantage with respect to other detection systems (i.e., colorimetric or even chemiluminescent with photographic detection) is the direct and rapid quantitative evaluation of the signal over a wide dynamic range. It can be used for the measurement of CL signals in microtiter plates, with the advantage of a one-step measurement of the emission from the whole plate (differently from conventional luminometers, which usually read the emission well by well or strip by strip). This may be an important advantage in analytical methods relying on the kinetic behavior of the CL emission. CL imaging is also used to detect BL and CL in whole organs or plants, and on any kind of surface. It should be noted that CL imaging devices do not possess the very wide dynamic range characteristic of photomultiplier tube-based instruments. However, this limitation can be