Methods and kits for detecting hemoglobin in test samples

Abstract

The present invention relates to methods of detecting hemoglobin in a test sample. These methods can be used to diagnose a subject suffering from a genetic disorder relating to hemoglobin metabolism, to determine the eligibility of a subject to be a blood donor, to determine the age of a stored blood sample or to identify a hemolyzed plasma sample. The present invention also relates to kits for use in the above described methods.

1 . A method of detecting hemoglobin in a test sample, the method comprising the steps of:
 a) adding at least one basic solution to a test sample;   b) adding an indicator solution to the test sample to generate a light signal, wherein the indicator solution comprises at least one acridinium compound;   wherein steps a) and b) can be performed in any order; and   c) measuring the light generated to detect the hemoglobin in the test sample.   
     
     
         2 . The method of  claim 1 , wherein the test sample is a non-biological forensic sample, stool, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, soil or a blood substitute. 
     
     
         3 . The method of  claim 1 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         4 . The method of  claim 1 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         5 . The method of  claim 1 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: 
         hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         6 . The method of  claim 1 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         7 . The method of  claim 1 , further comprising measuring the amount of hemoglobin in the test sample by relating the amount of light generated in the test sample by comparison to a standard curve for hemoglobin or to a reference standard. 
     
     
         8 . The method of  claim 7 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration. 
     
     
         9 . A method of detecting hemoglobin in a test sample, the method comprising the steps of:
 a) adding at least one basic solution to a test sample; and   b) measuring the current generated at at least one electrode to detect the hemoglobin in the test sample.   
     
     
         10 . The method of  claim 9 , wherein the test sample is a non-biological forensic sample, stool, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, soil or a blood substitute. 
     
     
         11 . The method of  claim 9 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         12 . A method of diagnosing a subject suffering from a genetic disorder relating to hemoglobin metabolism, the method comprising the steps of:
 a) adding at least one basic solution to a test sample obtained from a subject suspected of suffering from a genetic disorder relating to hemoglobin metabolism;   b) adding an indicator solution to the test sample to generate a light signal, wherein the indicator solution comprises at least one acridinium compound,   wherein steps a) and b) can be performed in any order;   c) quantifying the light generated to detect the hemoglobin in the test sample; and   d) determining the concentration of hemoglobin in the test sample based on the amount of light quantified in step c); and   e) comparing the concentration of hemoglobin in step (d) with a predetermined level, wherein if the concentration of hemoglobin determined in step (d) is lower or higher than the predetermined level, then a determination is made that the subject is suffering from a genetic disorder relating to hemoglobin metabolism.   
     
     
         13 . The method of  claim 12 , wherein the test sample is serum, plasma, whole blood, red blood cells and umbilical cord blood. 
     
     
         14 . The method of  claim 12 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         15 . The method of  claim 12 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         16 . The method of  claim 12 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         17 . The method of  claim 12 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         18 . The method of  claim 12 , further comprising measuring the amount of hemoglobin in the test sample by quantifying the amount of light generated in the test sample by comparison to a standard curve for hemoglobin or to a reference standard. 
     
     
         19 . The method of  claim 18 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration. 
     
     
         20 . The method of  claim 12 , wherein the genetic disorder relating to hemoglobin metabolism is anemia or β-thalassemia. 
     
     
         21 . A method of determining the eligibility of a subject to be a blood donor, the method comprising the steps of:
 a) adding at least one basic solution to a test sample obtained from a subject;   b) adding an indicator solution to the test sample to generate a light signal, wherein the indicator solution comprises at least one acridinium compound,   wherein steps a) and b) can be performed in any order;   c) quantifying the light generated to detect the hemoglobin in the test sample;   d) determining the concentration of hemoglobin in the test sample based on the amount of light quantified in step c); and   e) comparing the concentration of hemoglobin in step (d) with a predetermined level, wherein if the concentration of hemoglobin determined in step (d) is lower or higher than the predetermined level, then a determination is made that the subject is not eligible to be a blood donor.   
     
     
         22 . The method of  claim 21 , wherein the test sample is whole blood. 
     
     
         23 . The method of  claim 21 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         24 . The method of  claim 21 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         25 . The method of  claim 21 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         26 . The method of  claim 21 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         27 . The method of  claim 21 , further comprising measuring the amount of hemoglobin in the test sample by quantifying the amount of light generated in the test sample by comparison to a standard curve for hemoglobin or to a reference standard. 
     
     
         28 . The method of  claim 27 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration. 
     
     
         29 . A method of determining the age of a stored blood sample, the method comprising the steps of:
 a) adding at least one basic solution to a blood sample;   b) adding an indicator solution to the blood sample to generate a light signal, wherein the indicator solution comprises at least one acridinium compound,   wherein steps a) and b) can be performed in any order;   c) quantifying the light generated to detect the hemoglobin in the blood sample;   d) determining the concentration of hemoglobin in the blood sample based on the amount of light quantified in step c); and   e) comparing the concentration of hemoglobin in step (d) with at least one predetermined level, wherein if the concentration of hemoglobin determined in step (d) is lower then the predetermined level, then the plasma sample is determined to be an older blood sample.   
     
     
         30 . The method of  claim 29 , wherein the blood sample is whole blood, serum, plasma, platelets, red blood cells or umbilical cord blood. 
     
     
         31 . The method of  claim 29 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         32 . The method of  claim 29 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         33 . The method of  claim 29 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         34 . The method of  claim 29 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         35 . The method of  claim 29 , further comprising measuring the amount of hemoglobin in the blood sample by quantifying the amount of light generated in the blood sample by comparison to a standard curve for hemoglobin or to a reference standard. 
     
     
         36 . The method of  claim 29 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration. 
     
     
         37 . A method of identifying a hemolyzed serum or plasma sample, the method comprising the steps of:
 a) adding at least one basic solution to a serum or plasma sample;   b) adding an indicator solution to the serum or plasma sample to generate a light signal, wherein the indicator solution comprises at least one acridinium compound,   wherein steps a) and b) can be performed in any order;   c) quantifying the light generated to detect the hemoglobin in the serum or plasma sample;   d) determining the concentration of hemoglobin in the serum or plasma sample based on the amount of light quantified in step c); and   e) comparing the concentration of hemoglobin in step (d) with a predetermined level, wherein if the concentration of hemoglobin determined in step (d) is the same as or higher then the predetermined level, then the serum or plasma sample is determined to be hemolyzed and further wherein if the concentration of hemoglobin determined in step (d) is lower then the predetermined level, then the serum or plasma sample is determined not to be hemolyzed.   
     
     
         38 . The method of  claim 37 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         39 . The method of  claim 37 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         40 . The method of  claim 37 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         41 . The method of  claim 37 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         42 . The method of  claim 37 , further comprising measuring the amount of hemoglobin in the serum or plasma sample by quantifying the amount of light generated in the serum or plasma sample by comparison to a standard curve for hemoglobin or to a reference standard. 
     
     
         43 . The method of  claim 42 , wherein the standard curve is generated from solutions of hemoglobin of a known concentration. 
     
     
         44 . A kit for detecting hemoglobin in a test sample, the kit comprising:
 a. at least one basic solution;   b. at least one indicator solution, wherein the indicator solution comprises at least one acridinium compound; and   c. instructions for detecting hemoglobin in a test sample.   
     
     
         45 . The kit of  claim 44 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         46 . The kit of  claim 44 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         47 . The kit of  claim 44 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         48 . The kit of  claim 44 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         49 . A kit for detecting hemoglobin in a test sample, the kit comprising:
 a. at least one basic solution;   b. at least one electrode; and   c. instructions for detecting hemoglobin in a test sample.   
     
     
         50 . The kit of  claim 49 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         51 . A kit for diagnosing a subject suffering from a genetic disorder relating to hemoglobin metabolism, the kit comprising:
 a. at least one basic solution;   b. at least one indicator solution, wherein the indicator solution comprises at least one acridinium compound; and   c. instructions for diagnosing a subject suffering from a genetic disorder relating to hemoglobin metabolism.   
     
     
         52 . The kit of  claim 51 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         53 . The kit of  claim 51 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I:
 wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion.   
     
     
         54 . The kit of  claim 51 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         55 . The kit of  claim 51 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         56 . A kit for determining the eligibility of a subject to be a blood donor, the kit comprising:
 a. at least one basic solution;   b. at least one indicator solution, wherein the indicator solution comprises at least one acridinium compound; and   c. instructions for determining the eligibility of a subject to be a blood donor.   
     
     
         57 . The kit of  claim 56 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         58 . The kit of  claim 56 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         59 . The kit of  claim 56 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         60 . The kit of  claim 56 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         61 . A kit for determining the age of a stored blood sample, the kit comprising:
 a. at least one basic solution;   b. at least one indicator solution, wherein the indicator solution comprises at least one acridinium compound; and   c. instructions for determining the age of a blood sample.   
     
     
         62 . The kit of  claim 61 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         63 . The kit of  claim 61 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         64 . The kit of  claim 61 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         65 . The kit of  claim 61 , wherein the indicator solution further comprises at least one surfactant. 
     
     
         66 . A kit for identifying a hemolyzed serum or plasma sample, the kit comprising:
 a. at least one basic solution;   b. at least one indicator solution, wherein the indicator solution comprises at least one acridinium compound; and   c. instructions for identifying a hemolyzed serum or plasma sample.   
     
     
         67 . The kit of  claim 66 , wherein the basic solution is a solution having a pH of at least about 10. 
     
     
         68 . The kit of  claim 66 , wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present, X ⊖  is an anion. 
       
     
     
         69 . The kit of  claim 66 , wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         wherein R 3  through R 15  are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and 
         optionally, if present, X ⊖  is an anion. 
       
     
     
         70 . The kit of  claim 66 , wherein the indicator solution further comprises at least one surfactant.