Chemiluminescence in Analytical Chemistry
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Figure 11 Synthetic scheme for the synthesis of acridan ester GZ-11.
4.3 2-Chloro-Acridine-9-Carboxylic Acid
To a solution of oxalic chloride (5 g) in dichloromethane, a solution of diphenyl amine (5 g) in dichloromethane was added dropwise and refluxed for 30 min. The solution was concentrated (50%) and aluminum trichloride (8 g) added in portions. The mixture was refluxed for 45 min and the solvent evaporated. To this residue hydrochloric acid in ice water (1 M) was added and the red-colored precipitate filtered. The precipitate was dissolved in potassium hydroxide (10% in water), refluxed overnight, and poured into hydrochloric acid in ice water (5 M). The yellow acridine-9-carboxylic acid was filtered, washed with water, and dried.
4.4 Perfluoro-t-Butyl 2-Chloroacridine-9-Carboxylate
Acridine-9-carboxylic acid (1 g) was mixed with thionyl chloride (20 mL) and refluxed until a clear solution was obtained. The solution was concentrated and
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the excess thionyl chloride coevaporated with toluene. To the residue pyridine (20 mL), 4-dimethylaminopyridine (1 g) and an excess of perfluoro-tert-butanol were added. The mixture was stirred overnight at room temperature, poured into hydrochloric acid in ice water (1 M), and extracted with dichloromethane. The organic layer was washed with water, dried over MgSO4, and concentrated. The crude product was purified by flash chromatography (66% hexane/ethyl acetate).
1H NMR (CDCl3) δ 7.6–7.9 (m, 5H), 8.2–8.3 (m, 2H).
4.5Perfluoro-t-Butyl 2-Chloro-10-Methylacridinium-9- Carboxylate Trifluoromethanesulfonate
Acridine-9-carboxylic ester (100 mg) was dissolved in dichloromethane (5 mL). To this solution an excess of methyl trifluoromethanesulfonate was added. The mixture was stirred overnight at room temperature. Diethyl ether was added and the obtained precipitate was filtered, washed with diethyl ether, and dried.
1H NMR (acetone-d6) δ 5.3 (s, 3H), 8.2–9.3 (m, 7H).
4.6Perfluoro-t-Butyl 2-Chloro-10-Methylacridane-9- Carboxylate (GZ-11)
10-Methylacridinium-9-carboxylic ester trifluoromethanesulfonate (100 mg) was dissolved in dichloromethane (100 mL). To this solution perchloric acid (20 drops) and zinc powder (6 g) were added. The mixture was placed in an ultrasonic bath for 3 h and filtered. The solution was washed with water (1 ) and hydrochloric acid (2 , 0.1 M), dried over MgSO4, and concentrated.
1H NMR (CDCl3) δ 3.4 (s, 3H), 5.1 (s, 1H), 6.9–7.4 (m, 7H).
13C NMR (CDCl3) δ 163.9, 141.7, 140.8, 129.3, 128.9, 128.5, 125.7, 121.4, 121.2, 119.7, 117.8, 117.1, 114.0, 113.0, 49.5, 33.1
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Chemiluminescence and Bioluminescence in DNA Analysis
Masaaki Kai, Kazuko Ohta, Naotaka Kuroda, and
Kenichiro Nakashima
Nagasaki University, Nagasaki, Japan
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INTRODUCTION |
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ENZYMATIC CHEMILUMINESCENT AND |
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BIOLUMINESCENT DETECTIONS |
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Membrane Hybridization |
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Immobilized Hybridization |
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CHEMILUMINESCENT DETECTION OF ACRIDINIUM |
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ESTER–LABELED PROBE |
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DERIVATIZATION-BASED CHEMILUMINESCENT |
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DETECTION |
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DNA Quantification |
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Detection of Polydeoxyguanilic Acid (d(G)n)–Labeled Probe |
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CONCLUSIONS |
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1. INTRODUCTION
Over the last decade, various nonradiochemical methods utilizing chemiluminescent or bioluminescent reactions have been developed to increase the sensitivity and speed of detecting DNA probes or DNA itself. This has permitted the devel-
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Figure 1 HRP-catalyzed chemiluminescent reaction of luminol.
opment of powerful analytical techniques for obtaining information on gene structure and function.
Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2′- spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP).
Luminol is the most popular chemiluminescent compound. Chemiluminescent detection based on horseradish peroxidase (HRP)-catalyzed oxidation of luminol in the presence of hydrogen peroxide requires a halogenated phenol such as 4-iodophenol or 4-iodophenylboronic acid [1] as a potent enhancer (Fig. 1). This method is often called enhanced chemiluminescent detection. In the enhanced reaction, the light emission is increased over 100-fold, permitting detection of HRP at subfemtomole levels. The light has a maximum wavelength of 428 nm and can be captured with high efficiency by blue-light-sensitive X-ray film.
AMPPD is the best chemiluminescent substrate for detecting an ALPlabeled probe [2, 3]. The enhanced sensitivity of the chemiluminescence based on the reaction of AMPPD with ALP depends on the enzymatic reaction time (Fig. 2), because the slow kinetics of the signal decay result in the accumulation
Figure 2 ALP-catalyzed chemiluminescent reaction of AMPPD.
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of light (maximum wavelength of 470 nm). This protocol permits highly sensitive detection of enzyme molecules at subattomole levels [2].
Both ALP and HRP are commonly used as signal markers for chemiluminescent detection of complementary deoxyribonucleic acid (cDNA) probes. Recently, marine bacterial luciferase and firely luciferase have been utilized as the marker enzymes of cDNA probes [4, 5]. The light emission by reaction of luciferase with luciferin in the presence of ATP, magnesium(II) ion, and oxygen is called bioluminescence since this reaction is a naturally occurring chemiluminescent reaction (Fig. 3A) [6]. Luciferase also mediates an oxidative reaction of an aldehyde compound in the presence of the reduced type (FMNH2) of flavin mononucleotide (FMN), and then emits a strong and stable light (Fig. 3B) [6, 7]. These enzymatic chemiluminescent methods permit reliable and sensitive nonisotopic detection in immobilized hybridization assays, as well as in common membrane hybridization assays.
5-Bromo-4-chloro-3-indolyl (BCI) substrates can also be used with several commercially available marker enzymes, including ALP, β-D-galactosidase, and β-glucosidase (Fig. 4) [8]. The enzymes hydrolyze the BCI substrate, producing the corresponding chromogenic indigo dye and hydrogen peroxide stoichiometrically in the reaction mixture. Therefore, the enhanced luminol-HRP detection system can detect the enzyme used as a signal marker when high sensitivity is required in hybridization assays.
Acridinium esters have also been utilized for chemiluminescent detection of cDNA probes (Fig. 5) [9–11]. The hydrolysis rate is much faster when the ester is conjugated to single-stranded DNA, rather than to double-stranded DNA. This means that the chemiluminescence from unhybridized acridinium ester– labeled probe is rapidly lost, whereas the chemiluminescence from the hybridized probe is minimally affected. This permits discrimination between hybridized and unhybridized acridinium ester–labeled DNA probes without separation steps.
Figure 3 Luciferase-catalyzed bioluminescent reaction with (A) luciferin and (B) an aldehyde compound and FMNH2.
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Figure 4 Enzymatic reactions of BCI substrates and their chemiluminescent detection by the luminol reaction.
Therefore, chemiluminescent methods using an acridinium ester–labeled cDNA probe allow the discrimination of a mismatched DNA sequence in a homogeneous assay.
On the other hand, few reagents react directly with the target DNA for chemiluminescent detection. Recently, a unique chemical derivatization reagent, 3′,4′,5′-trimethoxyphenylglyoxal (TMPG), has been developed [12–15]. This reagent reacts specifically with guanine bases in nucleic acids to produce a chemiluminescent, fluorescent derivative quickly, under mild reaction conditions (Fig. 6). The derivative emits chemiluminescence (maximum wavelength of 510 nm) un-
Figure 5 Chemiluminescent reaction of acridinium ester and its hydrolysis.
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Figure 6 Derivatization and chemiluminescent reactions of guanine-containing compounds with TMPG.
der weakly alkaline conditions in the presence of dimethylformamide (DMF) at room temperature. The TMPG reaction can be used to detect DNA or polydeoxyguanylic acid (d(G)n), both on nylon membranes and in aqueous solution.
This chapter outlines the chemical principles for luminescent detection of target DNA in hybridization and quantitative assays that utilize the above-men- tioned chemiluminogenic and bioluminogenic reagents.
2.ENZYMATIC CHEMILUMINESCENT AND BIOLUMINESCENT DETECTIONS
Enzyme-labeled probes have been employed for sensitive chemiluminescent or bioluminescent detection of hybridized target DNA that is bound to a membrane or otherwise immobilized. For these assays, HRP, ALP, and luciferase have been used as the marker enzyme predominantly. These enzymes are far bulkier than isotopic labels, and are also less thermally stable. For labeling, a small molecule such as digoxigenin or biotin is employed to conjugate the enzymes to a DNA hybrid duplex by means of either bioaffinity interaction of avidin or immunochemical interaction of antidigoxigenin antibody [16]. The primary advantage of using biotin or digoxigenin probes is that they are suitable for indirect labeling of many unstable enzymes to cDNA probes.
2.1 Membrane Hybridization
Hybridization assays of membrane-bound DNA are important for characterizing or searching for cloned genes related to a genetic disease, and for identifying