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  Electrochemiluminescence from acridan compoundsRelated Patent Categories: Chemistry: Analytical And Immunological Testing, Peptide, Protein Or Amino AcidThe Patent Description & Claims data below is from USPTO Patent Application 20070059841. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority from Provisional U.S. Application No. 60/214,178, filed Jun. 26, 2000. FIELD OF THE INVENTION [0002] The present invention relates to the field of electrochemiluminescence which is the production of luminescence by an electrochemical reaction. In particular the present invention relates to methods of producing electrochemiluminescence from acridan compounds. The present electrochemiluminescent reaction can find use in assay methods for detecting analytes. The acridan compounds can be provided with a labeling group for linking to an analyte or analyte binding partner. BACKGROUND OF THE INVENTION [0003] Electrochemiluminescence (ECL) has received widespread attention during the previous decade, especially in the field of chemical analysis. It combines the well known sensitivity of chemiluminescence (CL) with the precise control over the time and position of light emitting reactions afforded by electrochemistry. As an alternative approach for conducting immunoassays and nucleotide assays it offers advantages such as increased sensitivity and precision, reduction in time and labor, and the elimination of radioisotopes. In order to exploit the full potential of this technology there is a requirement for new chemiluminescent compounds which can be initiated electrochemically. We show for the first time how CL can be triggered by electrochemical oxidation of acridan compounds. [0004] Prior to the present invention, ruthenium chelates and luminol derivatives were the only compounds that have been used in a significant number of analytical applications involving ECL. (J. K. Leland and M. J. Powell, J. Electrochem. Soc., 1990, 137, 3127; S. Sakura, Anal. Chim. Acta., 1992, 262, 49) Ruthenium chelates have been used for enzyme assays, but their most significant impact has been as labels for immunoassays and nucleotide assays. In these applications a combination of ECL and magnetic bead technology has found increasing use in pharmaceutical labs for high throughput screening. Luminol has also been used for enzyme assays and immunoassays. Light is emitted when electrochemically oxidized luminol reacts with hydrogen peroxide which allows the reaction to be coupled to oxidase enzymes such as glucose oxidase. (R. Wilson and A. P. F. Turner, Biosensors, 1997, 12, 277) The chemiluminescence reaction of luminol is also catalyzed by electrochemically oxidized ferrocenes (R. Wilson and D. J. Schiffrin, J. Electroanal. Chem., 1998, 448, 125) suggesting that these compounds could be used as labels in an ECL system resembling the one based on ruthenium chelates. [0005] Acridinium esters were discovered in 1964 and subsequently developed as labels for immunoassays and nucleotide assays. The chemiluminescence reaction mechanism of these compounds involves nucleophilic attack of a peroxide anion (HOO--) in alkaline solution on the 9-position of the acridinium nucleus followed by internal cyclization leading to the formation of a metastable dioxetanone intermediate. This spontaneously decarboxylates to give the singlet excited state of N-methylacridone, which emits blue light at 430 nm when it relaxes to the ground state. The chemiluminescence quantum yield is typically between 1 and 10%. The reaction is extremely rapid, but in the absence of peroxide other nucleophiles such as hydroxide ion can form an adduct (pseudo-base) with the 9-position of the acridinium nucleus. Formation of this intermediate precludes the formation of a dioxetanone intermediate and therefore no light is emitted unless pseudo-base formation is reversed by an acidic solution of hydrogen peroxide before adding a sodium hydroxide solution. [0006] Electrochemical triggering of the chemiluminescent reaction of an acridinium ester at a pH of 5.0 was developed in an attempt to simplify the conventional initiation procedure. (J. S. Littig and T. A. Neeman, Anal. Chem., 1992, 64, 1140-1144) This pH is not particularly useful for immunoassays and nucleotide assays. A solution of acridinium ester was injected into a flowing stream of pH 12 phosphate buffer and pumped into a flow cell. Chemiluminescence was triggered in the cell by reducing dissolved oxygen electrochemically. The conditions are a compromise between those required for chemiluminescence and oxygen reduction, and those necessary to avoid pseudo-base formation. It would also be necessary to control the concentration of dissolved oxygen to obtain precise results which cancels out the increase in simplicity obtained by initiating the chemiluminescent reaction electrochemically. These drawbacks are avoided when an acridan ester is used because the acridinium ester is produced in situ from a passive precursor. [0007] Recently a large number of acridans (reduced acridinium esters, thioesters and amides) based on the N-alkylacridancarboxylate nucleus, including DMC, have been made. (H. Akhavan-Tafti, et al., J. Org. Chem. 1998, 63, 930-937; H. Akhavan-Tafti et al., Clin. Chem. 1995, 41, 1368-1369) These acridan compounds are stable in the presence of hydrogen peroxide and do not form an inactive pseudo-base. Light emission can be triggered by enzymatically oxidizing the acridan with the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide and an enhancer such as p-iodophenol. HRP oxidizes the acridan to the corresponding acridinium ester, which in most cases is immediately subject to nucleophilic attack by the peroxide anion (HOO--) at the 9 position of the acridinium nucleus; the possibility of pseudo-base formation does not arise because peroxide is several orders of magnitude more nucleophilic than hydroxide. Nucleophilic attack on the acridinium ester results in the formation of a dioxetanone which decomposes to form the singlet excited state of N-methylacridone. This in turn relaxes to the ground state accompanied by the emission of intense blue light with a maximum wavelength of 430 nm. By using these compounds as a substrate for HRP it has been possible to detect as little as 0.1 amol of this enzyme in a 15 minute assay. [0008] Previous work on the electrochemistry of acridan which does not bear a carbonyl group at the 9-position demonstrated the oxidation by a mechanism in which the second oxidation step occurs in solution as a result of disproportionation between protonated and unprotonated radical intermediates. (P. Hapiot, J. Moiroux and J. M. Saveant, J. Am. Chem. Soc., 1990, 112, 1337) This reaction did not involve the production of chemiluminescence. [0009] Acridan compounds substituted with an oxidizable exocyclic double bond are disclosed in commonly assigned U.S. Pat. No. 5,922,558. These compounds are enzymatically oxidized by a peroxidase enzyme to produce visible light. The opposite terminus of the double bond bears two substituents, one being an ether or thioether-type group, the other being any of various groups such as ether or thioether-type groups, alkoxy, aryloxy, alkylthio, arylthio, trialkylsilyloxy, phosphoryloxy, acyloxy and acylthio groups. Compounds of this type having a phosphate salt group are also disclosed in commonly assigned U.S. Pat. No. 6,045,727 which describes their enzymatic reaction with phosphatase enzymes to produce chemiluminescence. SUMMARY OF THE INVENTION [0010] It is an object of the present invention to provide a method for producing chemiluminescence by an electrochemical reaction. It is a further object of the present invention to provide a method for producing chemiluminescence by the electrochemical reaction of acridan compounds in the presence of a peroxide, particularly hydrogen peroxide. A further object of the invention is to provide a method for conducting an assay of an analyte using an electrochemiluminescent reaction to produce light for detecting the analyte. GENERAL DESCRIPTION [0011] The present invention concerns the electrochemiluminescent oxidation of acridan derivatives. We have found that the electrochemical oxidation of acridan compounds in the presence of peroxide at neutral to alkaline pH results in the generation of visible luminescence above a certain minimum potential. The reaction adds to the small number of analytically useful electroluminescent reactions and can find use in assay methods for detecting analytes. For example, the acridan compound can be provided with a labeling group for linking to an analyte or analyte binding partner. [0012] A first group of acridan compounds useful in the practice of the present invention comprise acridan-carboxylic acid derivatives having the general formula: wherein R.sup.1 to R.sup.4 can be any of a variety of groups provided that they do not interfere with the production of chemiluminescence. The Z group is O,S, or NR wherein R can be any of a variety of groups but is preferably a sulfonyl group. [0013] Unlike art-known methods of generating luminescence electrochemically using an acridinium ester, the reaction does not involve the electrochemical generation of H.sub.2O.sub.2 or superoxide. The acridan compounds used in the present methods are significantly more stable that the corresponding acridinium compounds and should provide more robust labels. [0014] The electrochemiluminescence is generated by subjecting a solution of the acridan compound and peroxide to a positive potential above a certain threshold. The threshold value is easily determined by voltammetric scan or by measuring luminescence during a voltage sweep as described below. [0015] A second group of acridan compounds useful in the practice of the present invention comprise compounds having the general formula: wherein Z.sup.1 and Z.sup.2 are independently selected from O, S and NR atoms wherein R can be any of a variety of groups but is preferably a sulfonyl group, and wherein R.sup.1 to R.sup.4 and X can be any of a variety of groups provided that they do not interfere with the production of chemiluminescence. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 depicts a laminar flow cell used for electrochemiluminescence detection of acridans. [0017] FIG. 2 depicts two successive cyclic voltammograms of 50 .mu.M DMC in 10 mM Tris buffer with 0.1 M NaCl, 10 mM hydrogen peroxide, and 0.025% Tween-20. Scan rate 100 mV/s, the 1st scan contains peaks A and B; the 2nd scan contains peaks A, B and C. [0018] FIG. 3 is a graph showing the dependence of electrochemiluminescence on potential for 50 .mu.M DMC at pH 8.0, in 10 mM Tris buffer with 0.1 M NaCl, 10 mM hydrogen peroxide, 1 mM EDTA and 0.025% Tween-20 at a scan rate 10 mV/s. [0019] FIG. 4. (A) Light and (B) current transients for a potential step from 0 to 1 V for 5 nM DMC at pH 8.0, in 10 mM Tris buffer with 0.1 M NaCl, 10 mM hydrogen peroxide, 1 mM EDTA and 0.025% Tween-20. Continue reading... Full patent description for Electrochemiluminescence from acridan compounds Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Electrochemiluminescence from acridan compounds patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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