Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Chemiluminescent
Patent
1996-01-11
1998-11-17
Jones, W. Gary
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Chemiluminescent
435 6, 435 71, 435 792, 435287, 436164, 436516, 436518, 436807, G01N 2101, G01N 3353, G01N 2100, C12Q 168
Patent
active
058371940
DESCRIPTION:
BRIEF SUMMARY
This application is filed under 35 USC 371 as the national stage of International Application PCT/US94/04471, filed Apr. 25, 1994.
FIELD OF THE INVENTION
This invention relates to a method of and apparatus for detecting chemiluminescence from a multiplicity of discrete samples arrayed on a surface in a defined format, such as a matrix.
BACKGROUND OF THE INVENTION
Various assay systems including immunoassays, receptor-ligand assays and probe hybridization are known for detecting desired chemical and biological information. For example, probe hybridization is used to detect specific nucleic acid sequences in a sample and involves the formation of a base-paired duplex from two single stranded nucleic acid molecules. With probe hybridization, in the dot-blot technique, the nucleic acid may be bound to a flat matrix such as a nylon membrane or nitrocellulose paper and detected by reacting it against a labelled nucleic acid that is complementary to a portion of the bound nucleic acid. Such labeled nucleic acid probes can be used to detect target sequences by hybridization to DNA or RNA.
The most commonly used label in assay systems for nucleic acids is radioactive phosphorus (.sup.32 P). Although radioactive labels are widely used in research laboratories, well-known problems with such radioactive labels--e.g., short half-life, safety and disposal problems, and the necessity to perform long auto-radiographic exposures for detection--generally preclude their use in clinical and other settings. This has resulted in the development of a number of non-radioactive labels, and associated detection methods. For example, probes labelled with fluorescein and with enzymes such as alkaline phosphatase and peroxidase are in common use. However, utilization of these labels has been limited by their lack of sensitivity compared with .sup.32 P.
Recently, non-radioactive labeling and detection systems for nucleic acids have been introduced based on detection of chemiluminescence which have sensitivities closer to that of .sup.32 P. Noteworthy among such chemiluminescence detection systems are: Enhanced Chemiluminescence (ECL) from Amersham International; Flash.TM. from Stratagene Cloning Systems; and Digoxygenin (DIG) from Boehringer Mannheim. FIG. 1 illustrates the hybridization and chemiluminescence process for the DIG system.
As is well known, chemiluminescence is a form of luminescence resulting from chemical reactions. (The reaction leading to light emission in bioluminescence is similar but generally requires either oxygen or hydrogen peroxide as a reactant.) With either chemiluminescence or bioluminescence, light is emitted in the form of single photons. Other forms of luminescence are generally distinguished by the form of stimulus for the light emission, e.g., fluorescence, where light is emitted in response to an external stimuli, such as light, and scintillation, where multiple photons are produced by a single event, such as an electron emitted from a decaying radioactive atom.
Currently, in probe hybridization assays, chemiluminescence is most often detected by exposing the matrix containing the sample plus hybridized labelled probe to X-ray film for a period of 10 min. to several hours, depending on the required sensitivity. Such autoradiography does not permit direct quantification of chemiluminescence, but densitometry may be applied to the x-ray film to obtain a quantitative measure of limited range.
An alternative approach is to use photodetectors to read the chemiluminescence. A common instrument having such detectors is the liquid scintillation counter. Such counters normally operate in a coincidence mode, in which a pair of oppositely disposed photo-multiplier tubes must both detect an event for the event to be registered as a multi-photon event characteristic of scintillation. However, by turning off one of such oppositely-disposed photo-multiplier tubes, the machine can count single photon events. In this manner, a liquid scintillation counter could be used to detect and quantify chemiluminescence, e
REFERENCES:
patent: 4283490 (1981-08-01), Plakas
patent: 4298796 (1981-11-01), Warner et al.
patent: 5294795 (1994-03-01), Lehtinen et al.
patent: 5306617 (1994-04-01), Uchida et al.
Anderson Larry J.
Potter Colin G.
Jones W. Gary
Tran Paul B.
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