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APPLICATIONS |
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Enzyme Substrates (A Review) |
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Enzyme conjugates are used in enzyme-linked immunosorbent assays, blotting techniques, in-situ hybridization, cytometry and histometry. Most frequently Horseradish Peroxidase, Alkaline Phosphatase and b-Galactosidase enzymes are used because of their high turnover rate, stability, ease of conjugation and relatively low cost. Michigan Diagnostic LLC actively involved developing better substrate for different enzymes. These enzyme substrates are safer, more sensitive and faster compared to radioactive methods. These substrate are of three types. |
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1. Chromogenic |
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2. Fluorogenic |
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3. Lumigenic |
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Chromogenic Substrates: |
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These substrates produced color in the presence of suitable enzymes. When a solution of p-nitro phenyl phosphate in tris-buffer is treated with small amount of Alkaline Phosphatase, it produces bright yellow color and then can be monitor at 405nm in a colorimetric spectrometer. The reaction can be shown as: |
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Alkaline Phosphatase enzyme cleaves oxygen and phosphorous bond and produce yellow color anion of p-nitro phenol, which is stable at pH 9.5 to 10.2. For chromogenic substrates, higher concentration enzyme (1.8x108 molecules of Alkaline Phosphatase) is required. |
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Fluorogenic Substrates: |
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Fluorogenic substrates are several orders of magnitudes more sensitive than chromogenic substrates. Fluorescein diphosphate (FDP) is an excellent substrate for alkaline phosphatase in enzyme-linked immunoabsorbent assays. The high pH used to monitor alkaline phosphatase activity is advantageous since it also enhance fluorescence’s fluorescence. Moreover, fluorescein diphosphate is colorless and nonfluorescent, and its hydrolysis product, fluorescein, has superior spectral properties (quantum yield=0.9), which should make FDP the most sensitive fluorogenic substrates available for detecting alkaline phosphatase activity. |
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Fluorogenic technique can detect 1.8x105 molecules of alkaline phosphatase. |
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Lumigenic Substrates: |
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Chemiluminescence is defined as the production of light chemically. Exothermic chemical reactions release energy during the course of the reaction. The basic mechanism for light production involves thermal or catalyzed decomposition of high-energy compounds to produce the reaction product in singlet or triplet state. The emission of light from the excited species in singlet state is known as chemiluminescence. |
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The most familiar example of chemiluminescence is the emission of light by the firefly and has a quantum efficiency of 88%. These biological processes are catalyzed by photo-proteins or enzymes called luciferase which activate a substrate (luciferin) and molecular oxygen to produce an intermediate peroxide. The luciferin is converted in the presence of ATP and oxygen to four-membered ring peroxide which decomposes to generate the observed yellow-green luminescence. In 1966, McCapra suggested the intermediate of a dioxetanone as the key high-energy intermediate in the firefly system. |
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White and coworkers described the effects of various substituents on in vitro firefly bioluminescence. A series of luciferins with different substituents X were prepared. White noted that both the synthetic luciferin with X = NH2 and the natural luciferin (X = O-) gave light while the reactions with the methyl ether and amide derivatives were dark. |
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Radioactive isotope labels are widely used in immunoassays. Antigen and antibodies can be labeled with several radioactive isotopes. Because the radiation can penetrate through media, this gives rise to disposal problems, special laboratory requirements and maximum doses for use of these isotopes. Short-lived nature limits the shelf-life of labeled antigen and antibodies. Other disadvantages include the long time required for scintillation counting, the high production cost and the difficulties in the purification of radioactive antigen and antibodies. Because of the disadvantages large efforts are being put into the search for alternate labels. |
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The use of chemilunescence material show great promise as superior to radioactive methods in a variety of application in molecular biology, immunology and biotechnology. Luminol and isoluminol have been used for steroids (cortisol, testosterone, 19-nortestosterone, progesterone, estradiol, and gonadal steroids), thyroxine and digoxin in chemiluminescent assays. Protein chemiluminescent immunoassays have been developed for human IgG, rabbit IgG and alphafetoprotein. There are a number of advantages of using chemiluminescent labels. These substances present no known hazard to the environment. Generally, chemiluminescent reagents are inexpensive and readily available. The equipment required for the measurement of luminescence is relatively inexpensive. Since chemiluminescence is an emission process, response is usually linearly proportional to concentration. The reaction of luminol takes place in aqueous solution with hydrogen peroxide and a supplemental oxidant including ferricyanide, hypochlorite, per-sulfate, or the hydroxyl radical generated from hydrogen peroxide and metal derivative such as hemin. Chemiluminescent reaction also takes place with oxygen and a strong base in a dipolar aprotic solvent such as dimethyl sulfoxide. Under both conditions quantum yield is about .01. |
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1,2-Dioxetanes that can be synthesized on photo-oxidation of electron rich carbon-carbon double bond in the presence of Rose-Bengal and oxygen at low temperature are high-energy storage organic compounds. These high-energy compounds when decomposed produced light without generation of heat. Suitably derivatised adamantylideneadamantyl 1,2-dioxetanes were used as labels in thermo luminescent immunoassays by Hummelen et al in 1987 and is the first important application of 1,2-dioxetane to clinical analysis. |
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Now technology is well known for the use of 1,2-dioxetanes as a substrate for different enzymes. The advent of sensitive chemiluminescent detection of antigen, antibodies and other biological materials has facilitated the development of nonradioactive procedures in immunoassays. Recently the use of 1,2-dioxetanes molecules is increasing exponentially both in research and hospitals. The stable 1,2-dioxetanes are very convenient for storage at room temperature with a half-life of several years and can be modified as enzyme substrates to use in aqueous system. |
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The stabilized 1, 2-dioxetane of present invention can be shown as: |
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Upon enzymatic dephosphorylation, [(4-Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane-3,3-tricyclo[7.3.1,0 2,7] tridec-2,7-ene] disodium salt (G-8) becomes unstable and decompose by emiting light. The rate of light emission is proportional to the concentration of enzyme present. The anion produced upon dephosphorylation is moderately stable and accumulates before decomposition. This results in a kinetic delay that precedes the steady state of light emission. A constant rate of light emission is attained in approximately 30 minutes in a buffered solution at pH 9.5 to 10.2 at 30oC. |
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These novel 1,2-dioxetanes have overall superiority compared to the 1,2-dioxtanes known in the literature. A comparative light out put of [(4-Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane-3,3-tricyclo[7.3.1,0 2,7] tridec-2,7-ene] disodium salt ( G-8) and [(4 -Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane- 3,2’-(5-chloroadamantane)] disodium salt (CDP star) can be shown as: |
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Potential use of chemiluminescent substrates: |
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Chemiluminescent and bioluminescent reactions are becoming increasing important in ultra sensitive assays for clinically significant substance i.e. hormones, cancer markers, viral antibodies and in the study of biological process such as phagocytes. Two notable recent development are the high sensitivity assays for HRP and Alkaline Phosphatase labels. The peroxidase assay is based on enhanced chemiluminescent luminol reactions and the alkaline phosphatase assay utilizes a stabilized chemiluminescent 1, 2-dioxetane phosphate substrate which undergoes an enzyme catalyzed decomposition to produce a characteristic chemiluminescence emission. |
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Enzyme labels provide a sensitive, nonisotopic detection method due to their inherent catalytic amplification. Sensitivity can be further improved by combining an enzyme label with an ultra sensitive luminescent detection method for the detection of enzyme. |
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Alkaline phosphatase exhibits an unusually high degree of heat stability. The crude enzyme, extract from E. Coli, shows no loss of activity when heated at 85oC for 30 minutes. The purified enzyme, in the presence of magnesium, can be heated for 8 minutes at 95oC with retention of 50% of the activity. In addition, alkaline phosphatase is remarkable stable in low pH environments. The enzyme can be stored at pH 2.0 for 3 days at 4oC with retention of up to 80% of its activity. This unusual resistance to environmental conditions and its thermal stability make alkaline phosphatase the enzyme of choice as a label for DNA detection. |
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Immunoassays: |
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The new series of stabilized chemiluminescent 1,2-dioxetanes can detect 10-23 moles of alkaline phosphatase. |
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Materials and Methods: |
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Stabilized 1,2-dioxetanes: The method of preparation of [(4 -Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane-3,3-tricyclo[7.3.1,0 2,7] tridec-2,7-ene] disodium salt can be found in PCT/US99/20590. Into 1000mL of a 1 M solution of the tris-buffer (pH 9.5 to 9.7) was dissolved 0.2 parts of [(4 -methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane-3,3-tricyclo[7.3.1,02,7] tridec-2,7-ene], disodium salt. |
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Enzyme Diluent: The enzyme diluent was prepared by dissolving Tris (hydroxymethyl)aminomethane, sodium chloride, potassium chloride, zinc chloride, alcohols, and proteins in deionized water and the pH of the buffer was adjusted to 8.0 to 8.5. |
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Enhancers: Water-soluble polymers, partially water-soluble polymers and waster-insoluble or polymers soluble in organic solvent are well known in the literature and prepared as reported in US Patent # 2,780,604; 3,178,396; 3,770,439; 3,898,088; 4,308,335; 4,340,522; 4,424,326; 4,563,411; and 3,239,519 and new polymers were synthesized as reported in US patent application # 09/883,586 and publication # 2002-001350-A1. Polyvinylbenzyltrioctylphosphonium chloride (0.7g) was dissolved in 100mL of dimethylsulfoxide. To 100mL of the 1,2-dioxetane solution 2mL of the polymer solution was added and mixed properly. |
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Three different sources of alkaline phosphatase were used (1). Sigma Chemical Company, Catalog # P7923, lot # 128H1210 and 6,510 U / mg of protein, (2). Worthington Biochemical Corporation, 2000U / vial, 2,800 U / mg protein and lot # 69k3248, and (3). Roche Diagnostic Corporation (Boehringer Mannheim ), 2000 U / mg protein and lot # 85382422. |
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Each of these enzymes were diluted in the above defined diluent in which the enzyme is stable for an extended period of time at room temperature. For each source of enzyme, the concentration of alkaline phosphatase was adjusted to 0.27 mg/ml of the buffer. This solution contains 2.7 parts of alkaline phosphatase in 10,000 parts of modified buffer (dilution #1). Then 100 ml of the dilution #1 of alkaline phosphatase solution is further diluted in 900 ml of the buffer to yield dilution #2. This solution contains 2.7 parts of alkaline phosphatase in 100,000 parts of buffer (dilution #2). Next 100ml of this dilution #2 was added to 900 ml of the enzyme diluent to form dilution #3. The dilution was continued to prepare dilutions #4, #5, #6, #7, #8, #9, #10, #11. #12, #13 and #14. Dilution # 14 has the enzyme concentration of 2.7x10-14 mg/ ml or 2.7x10-17g/ml of buffer. In other words, 10 ml of dilution # 14 has 2.7x10-19 g of alkaline phosphatase. In the kinetic experiments, 10 ml of enzyme was used in 200 ml of the 1,2-dioxetane sample from dilutions #14, #13, #12 and #11. |
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Two hundred micro-liters (0.200mL) of 1,2-dioxetane in the buffer was transferred into a tube (75x12 mm) and background luminescence was recorded in a Monolight 2010 single tube luminometer. Then, 10 µl of each enzyme, as dilution #14, was added and the output light intensity was recorded at a 60 second of interval of time. The results are shown herewith: |
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Fig.1 is a graph showing a comparison of the chemiluminescence of each enzyme #14 dilution. Each dilution contains 0.27 attogram or at least a single molecule of alkaline phosphatase when 10ml of the enzyme was used in 200ml of the 1, 2-dioxetane sample in tris-buffer. |
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Fig. 2 is a graph showing a comparison of the chemiluminescence of each of the three #13 diluted enzymes. Each dilution #13 has at least 10 molecules of alkaline phosphatase. |
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Fig. 3 is a graph showing a comparison of the chemiluminescence of each of the three #12 diluted enzymes. Each dilution #12 has at least 100 molecules of alkaline phosphatase. |
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Fig. 4a are graphs of the chemiluminesce of the Sigma enzyme diluted to dilutions #13 and #14. The enzyme dilutions #13, and #14 have at least 1, and 10 molecules of alkaline phosphatase, respectively. |
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Fig. 4b are graphs of the chemiluminescence of the Sigma enzyme diluted to dilutions #11 and #12. The enzyme dilutions #11, and #12 have at least 100 and 1000 molecules of alkaline phosphatase, respectively. |
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The use of enzyme-linked immunoassays and DNA probes is finding increasing use in fields such as life science research, medical diagnostics, DNA profiling and microbe screening. Chemilunescence detection with triggerable 1, 2-dioxetanes offer much greater sensitivity than colorimetric or fluorimetric methods and eliminates the hazards of exposure, limited shelf-life and disposal problems associated with radioactive materials. |
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Results and Discussion: |
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The molecular weight of alkaline phosphatase enzyme is considered as being between 140,000 to 160,000, as reported in the literature and depending on the amounts of sugar units it contains. Using an assumed molecular wieght of 160,000 and divided by the Avogadro's number which is 6.022x10^23, the weight of one molecule of alkaline phosphatase can be calculated as at least 2.66x10^-19g or 0.266 attogram. The diluted samples of enzyme (#14) at least more than one molecule of alkaline phosphatase is present in 10 ml of the enzyme sample. Table 1 shows the ratio of background and signal obtained by the enzyme #14 is at least 5. |
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The results showin in Figure 1,2,3, and 4 shows the detection of alkaline phosphatase at attogram level and the results are confirmed from the three source of enzymes.The results shown in table 1 are obtained when different amounts of alkaline phosphatase are treated with 1, 2-dioxetane having p -electrons in the ring and an enhancer. |
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The present research results shows the detection of alkaline phosphatase enzyme at attogram level or single molecule detection in aqueous buffers utilizing a chemiluminescent system which comprises (1) a stable 1,2-dioxetane derived from a spiro-fused ketone with or without p -electrons in the ring or with carbon-carbon double bond(s) in the spiro-fused ring, (2) an polymeric enhancer or enhancers which is either, a water-insoluble, soluble in organic media, partially water-soluble, water-soluble, the polymer being derived from polymeric vinylbenzyl chloride and a trisubstituted amine or trisubstituted phosphine with or without fluorescent molecules, and (3) an enzyme diluent which stabilizes the enzyme during the reaction. |
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Thus, the present system for the detection of alkaline phosphatase at very low level may be diagrammatically illustrated as: |
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This is the first example to detect attogram level of alkaline phosphatase and the technique can be used in the early stage of detection of diseases. |
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Southern Blotting: |
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The southern blot technique permit the detection of a DNA sequence relative to restriction enzyme sites. Southern blotting requires electrophoretic separation followed by a transfer of resulting DNA fragments to a membrane. The degree of hybridization of a complementary probe to the DNA target in a sample is a measure of the amount of the specific target sequence in the sample. Detection of DNA is most often performed with radio isotopes such as 32P and 35S. Nonisotopic DNA detection techniques have a principally incorporated alkaline phosphatase as the preferred label due to its thermal stability and high turn over rate. In such system, detection of a desired nucleic acid sequence is limited by the ability to measure a low concentration of alkaline phosphatase. |
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Northern Blotting: |
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Chemiluminescent material is used for a rapid detection of membrane-bound RNA. It requires no special equipment and results can be conveniently imaged on instant photographic film or X-ray film with 5-20 minutes of exposure. |
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Western Blotting: |
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Western blots for detection for quantitation of proteins employ several different different colorimetric reagents for the detection of signal. Under usual circumstances as little as a few nanogram of proteins can be detected with relative ease. While analyzing whole bacterial cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting to detect altered forms of F-plasmid DNA replication protein Rep E, it is difficult to get a consistent signal without overloading the gel. Chemiluminescent substrates are better to obtain several fold gain in intensity to the signal that is detected on a standard x-ray film. |
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Plaque/Colony Lifts: |
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Easily detect biotin or digoxigenin-labeled DNA in minutes. |
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DNA Sequencing: |
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The ability to determine DNA nucleotide sequence rapidly and accurately has become increasingly important, as efforts have commenced to determine the sequence of the large genomes of humans and other higher organisms. Both the chemical and enzymatic sequencing methos yield isotopically labeled sequence ladders (composed of nested DNA fragments terminating at an A, F, C, or T) that are imaged directly on x-ray film after. |
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