High Sensitivity Detection of Bacterial Endospores via Tb Photoluminescence Enhancement

Detecting bacterial endospores is a critical challenge to bioanalytical chemistry, since a number of serious diseases and health problems are caused by members of the sporeforming genera Bacillus and Clostridium. We have developed a highly sensitive method for their detection and have demonstrated detection limits of less than 5000 CFU/ml. Our method is based on the presence of a marker compound in bacterial endospores, dipicolinic acid (dpa). When complexed with Tb and excited in the UV, the dpa enhances the photoluminescence emission of Tb by several orders of magnitude. We have investigated the potential for interference from other biological materials and chemicals and found that nothing other than bacterial endospores will give us a positive response to this test. Our investigation also showed that the presence of phosphate or organophosphate ions will reduce the observed signals. We have been able to overcome this problem through the addition of AlCl3. The results of our interference studies and phosphate studies will be presented. Since only 10% or less of the dpa is released when the endospores are suspended in aqueous buffer, we have also examined methods for enhancing the release of dpa. Our results from both mechanical and chemical methods to enhance dpa release will be presented. The best we have achieved is a 20-fold increase in dpa release from B. globigii endospores in 2 minutes through the addition of dodecylamine and heating to 80°C.