基因表現與分析

　Tropix's Substrate (Chemiluminescent Substrates & Chemiluminescence Enhancers)

　Principles of Enzyme-Activated Chemiluminescence
1,2-Dioxetane substrates emit visible light upon enzyme-catalyzed decomposition. Chemiluminescent detection of bio-analytes with 1,2-dioxetane enzyme substrates is extremely sensitive as a result of low background luminescence coupled with high-intensity light output due to enzyme catalysis. The energy for light emission is generated internally upon dioxetane decomposition. By comparison, fluorescence requires an external light source for excitation energy, which must be filtered to discriminate the fluorescent signal, This limits the sensitivity and introduces complexity into the determination.

　 Glow Kinetics of 1,2-Dioxetanes
The decomposition of CDP-Star ® substrate is shown (see opposite page, right). Upon dephosphorylation of the substrate by alkaline phosphatase, a metastable phenolate anion intermediate is formed that decomposes and emits light with a maximum intensity at a wavelength of 475nm. A delay in reaching maximum light intensity results, the length of which depends upon anion structure and the surrounding environment. Film or simple instrumentation may be used to quantitate the chemiluminescent signal that is produced as a steady glow arising from the reaction kinetics of the system.

　 1,2-Dioxetane Chemiluminescent Substrates
Tropix 1,2-dioxetane chemiluminescent substrates enable extremely sensitive detection of biomolecules by producing visible light that is detected with film or instrumentation. Applied Biosystems offers several different enzyme-activated substrates, including CDP-Star ® and CSPD® substrates for alkaline phosphatase; Galacton®, Galacton-Plus®, and Galacton-Star ® substrates for β-galactosidase; Glucuron® substrate for β-glucuronidase; Glucon™ substrate for β-glucosidase; and NA-Star™ substrate for neuraminidase. Use of these reagents in immunoassays, enzyme assays, reporter gene assays, membrane-based protein detection, and nucleic acid detection on membranes or in single tubes or microwells offers substantial benefits compared to colorimetric, fluorescent, or isotopic detection.

　 Advantages of 1,2-Dioxetane Substrates
Applied Biosystems offers a wide selection of 1,2-dioxetane enzyme substrates that meet the challenging demands of a broad variety of applications. 1,2-Dioxetane substrates are non-isotopic and provide high intensity signal, low background, wide dynamic range, rapid results, and are compatible with multiple assay formats under physiologically relevant conditions. The high quality, purity, and lot-to-lot consistency of Tropix substrates enable excellent reproducibility, In membrane-based assays, multiple film exposures can be acquired with standard X-ray film for over 24 hours following substrate addition.

　 Principles of Enzyme-Activated Chemiluminescence
1,2-Dioxetane substrates emit visible light upon enzyme-catalyzed decomposition. Chemiluminescent detection of bio-analytes with 1,2-dioxetane enzyme substrates is extremely sensitive as a result of low background luminescence coupled with high-intensity light output due to enzyme catalysis. The energy for light emission is generated internally upon dioxetane decomposition. By comparison, fluorescence requires an external light source for excitation energy, which must be filtered to discriminate the fluorescent signal, This limits the sensitivity and introduces complexity into the determination.

　Applications

Alkaline Phosphatase Substrates
使CDP-Star ® and CSPD® substrates are used in both solution and membrane-based detection of alkaline phosphatase or alkaline phosphatase conjugates as superior alternatives to radioisotopic, colorimetric, or fluorimetric methods. For immunoassays and DNA probe assays, 1,2-dioxetane substrates improve the sensitivity (Albrecht et al., 1991; Bronstein et al., 1989; Martin et al., 1995; Nishizono et al., 1990) and generate results faster than radioimmunoassay and colorimetric detection methods. Chemiluminescent detection of biomolecules labeled directly or indirectly with alkaline phosphatase (AP) is performed on membranes for Southern, northern, and western blotting, and DNA sequencing (Bronstein et al., 1990; Bronstein et al., 19992; Martin et al., 1991). These substrates are also suitable for the detection of placental alkaline phosphatase (PLAP) and secreted placental alkaline phosphatase (SEAP) in reporter gene assays (Henthorn et al., 1988; O' Connor et al., 1994; Bronstein et al., 1994a).
β-Galactosidase Substrates
Galacton®, Galacton-Plus®, and Galacton-Star ® substrates are utilized for quantitating β-galactosidase in reporter gene assays. The chemiluminescent assay for β-galactosidase exhibits over three orders of magnitude greater sensitivity than colorimetric assays.

β-Glucuronidase Substrate
Glucuron® is a highly sensitive substrate for quantitating β-glucuronidase in reporter gene assays using the bacterial β-glucuronidase (GUS) gene system. Chemiluminescent detection with Glucuron® exhibits greater sensitivity compared to fluorescence detection. Reporter gene assays employing Glucuron® are simple and convenient to perform.


β-Glucosidase Substrate
Glucon™ substrate is utilized for highly sensitive detection of β-glucosidase. The use of Glucon™ provides researchers with another quantitative tool for the emerging widespread use of glycosidic enzymes in environmental and biomedical testing, clinical evaluation, toxicology, and pharmaceutical screening.

Neuraminidase Substrate
NA-Star™ Substrate was designed for highly sensitive chemiluminescent detection of viral neuraminidase. NA-Star™ is a sensitive chemiluminescent replacement for fluorescent neuraminidase substrates.

Comparison of Chemiluminescent Systems
An important feature of Tropix alkaline phosphatase substrates is the long-lived signal, especially on membranes (see figures to the right). The chemiluminescent signal from CSPD® and CDP-Star ® substrate may persist for up to several days on nylon membrane. Since film exposure times range from minutes to several hours, multiple images may be acquired. Varying the film exposure time enables the user to optimize signal-to-noise. Other chemiluminescent system, such as enhanced luminol, generate shorter-lived signals, making multiple film exposures difficult.
In solution assays (such as in an immunoassay), the kinetics of CSPD® and CDP-Star ® substrates are more similar. Both substrates exhibit peak light emission within 30 minutes after adding substrate solution to a sample. Once the maximum signal has been reached, it will be maintained as long as substrate is available (at least 60-90 minutes).

Substrate Comparison：
CDP-Star ® Compared to CSPD®

CDP-Star ® chemiluminescent substrate combines high intensity with rapid kinetics of light emission. This feature, coupled with up to 10-fold higher signal intensity compared to CSPD® substrate, makes CDP-Star ® substrate the ideal choice when rapid exposures are needed. Furthermore, CDP-Star ® substrate is recommended in applications such as DNA fingerprinting with AP-labeled oligonucleotides which usually require long film exposures when utilizing radioactive probes. CDP-Star ® can be used in conjunction with Nitro-Block-IITM enhancer for rapid results on nitrocellulose membranes (see Chemiluminescence Enhancers, pape14).
CDP-Star ® substrate also produces a higher signal in solution assays (see figure). CDP-Star ® substrate with either Sapphire-IITM or Emerald-IITM enhancer produces a signal that is nearly fivefold higher than the signal produced by CSPD® substrate with enhancer. If maximum sensitivity is a requirement for your assays, CDP-Star ® solutions will provide the greatest sensitivity.

  


相關技術文件

　Reference:

  • Albrecht, S., H. Ehle, K. Schollberg, R. Bublitz, and A. Horn. 1991. Chemiluminescent enzyme immunoassay of human growth hormone based on adamantly dioxetane phenyl phosphate substrate, p. 115-118. In P. Stanley and L.J. Kricka (eds.), Bioluminescence and Chemiluminescence: Current Status, John Wiley, Chichester, England.
  • Bronstein, I., B. Edwards, and J.C. Voyta. 1989. 1,2-dioxetanes: novel chemilumi-nescent enzyme substrates: Applications to immunoassays. J. Biolum. Chemilum. 4:99-111.
  • Bronstein, I., R.R. Juo, J.C. Voyta, and B. Edwards. 1991. Novel chemiluminescent adamanthl 1,2-dioxetane enzyme substrates, p. 73-82. In P. Stanley and L.J. Kricka (eds.), Bioluminescence and Chemiluminescence: Current Status, John Wiley, Chichester, England.
  • Bronstein, I., J.C. Voyta, K.G. Lazzari, O.J. Murphy, B.Edwards, and L.J. Kricka. 1990. Rapid and sensitive detection of DNA in Southern blots with chemiluminescence. Bio Techniques 8:310-313.
  • Bronstein, I., J.C. Voyta, O.J. Murphy, L. Bresnick, and L.J. Kricka. 1992. Improvedchemiluminescent western blotting procedure. BioTechniques 12:748-753.
  • Bronstein, I., J.C. Voyta, G.H.G. Thorpe, L.J. Kricka, and G. Armstrong. 1989. Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin. Clin. Chem. 35:1, 441-1,446.
  • Bronstein, I., J.J. Fotrin, J.C. Voyta, R.R. Juo, B. Edwards, C.E.M. Olesen, N. Lijam, and L.J. Kricka. 1994a. Chemiluminescent reporter gene assays: Sensitive detection of the GUS and SEAP gene products. BioTechniques 17:172-178.
  • Bronstein, I., C.E.M. Olesen, C. Martin, G. Schneider, B. Edwards, A. Sparks, and J.C. Voyta. 1994b. Chemiluminescent detection of DNA and protin with CDP and CDP-Star 1,2-dioxetane enzyme substrates, p. 269-272. In A.K. Campbell, et al. (eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects, John Wiley, Chichester, England.
  • Edwards, B., A. Sparks, J.C. Voyta, and I. Bronstein. 1994. New chemiluminescent dioxetane enzyme substrates, p. 56-59. In A.K. Campbell, et al.(eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects, John Wiley, Chichester, England.
  • Henthorn, P., P. Zervos, M. Raducha, H. Harris, and T. Kadesch. 1988. Expression of ahuman placental alkaline phospatase gene in transfected cells: Use as a reporter for studies of gene expression. Proc. Natl. Acad. Sci.USA 85:6, 342-6,346.
  • Martin, C., L. Bresnick, R.R. Juo,, J.C. Voyta, and I. Bronstein. 1991. Improved chemiluminescent DNA sequencing. BioTechniques 11:110-113.
  • Martin, C., L. Butler, and I. Bronstein. 1995. Quantitation of PCR products with chemiluminescence. BioTechniques 18:908-913.
  • Nishizono, I., L. Lida, N. Suzuki, H. Kawada, H. Murakami, Y. Ashihara, and M. Okada. 1991. Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers. Clin. Chem. 37:1,639-1,644.
  • O’Connor, K.L. and L.A. Culp. 1994. Quantitation of two histochemical markers in the same extract using chemiluminescent substrates. BioTechniques 17:502-509.

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