A technique for detecting bacterial endospores via luminescence affords a sensitivity much greater than that of a prior luminescence-based technique from which it is derived. The advantage of luminescence-based detection is that the entire preparation-and-detection process takes only minutes, whereas a conventional process of culturing cells, staining cells, and examining cells under a microscope can take hours or days. The present technique could be especially useful for environmental monitoring of pathogenic bacterial endospores.

A Single DPA Molecule can enter the coordination sphere of a Tb3+/crown-ether complex. The DPA absorbs ultraviolet light, some of the energy of the absorbed light is transferred into the complex, and photons containing some of the transferred energy are emitted. These photons are detected to obtain a measure of the concentration of spores from which the DPA molecules were released.
In the prior technique, one prepares a sample by adding a great excess of TbCl3 to an aqueous suspension that contains bacterial endospores. The Tb3+ ions formed by the dissolution of TbCl3 interact with H2O to form [Tb(H2O)9]3+ complexes. A typical bacterial endospore contains between 2 and 15 weight percent of dipicolinic acid (DPA). The [Tb(H2O)9]3+ reacts with DPA released from the spore casing to generate a monochelate [Tb(DPA)(H2O)6]+ complex. Particles are removed from the suspension by use of a 0.22-μm filter. Under ultraviolet illumination at the wavelength of maximum absorption in DPA, the [Tb(DPA)(H2O)6]+ luminesces with an intensity greater than that of [Tb(H2O)9]3+. The intensity of luminescence can be measured and used to estimate the concentration of spores by reference to a calibration curve of intensities measured previously at known spore concentrations.

The main limitation on sensitivity of detection arises from the need for the great excess of the concentration of terbium over that of DPA. Excess terbium ensures that out of three Tb chelates that can exist in equilibrium, the one that predominates is the desired monochelate [Tb(DPA)(H2O)6]+. The photophysical properties (e.g., quantum yield and luminescence lifetime) of the other chelates are such that if allowed to remain in significant quantities, they would detract from the measurements. Unfortunately, the great excess of Tb needed for forming monochelates also leads to a large, undesirable background luminescence attributable to unchelated Tb3+, with consequent adverse effect on detection. Moreover, coordinated water molecules contribute undesired efficient nonradiative decay pathways that drastically reduce the quantum yield of luminescence, with consequent further adverse effect on detection.

The present improved technique is based on the idea that if it were possible, it would be preferable to have Tb in slight excess to reduce the background luminescence attributable to unchelated Tb3+ while simultaneously preventing the equilibrium formation of the undesired chelates and eliminating coordinated water. In this technique, the analysis reagent is a supramolecular complex that comprises a central lanthanide ion (which could be Tb3+) caged by a crown ether. The six oxygen atoms in the crown ether occupy most of the coordination sites of a lanthanide ion. A light-harvesting DPA molecule can enter this complex at the remaining coordination sites and be detected by luminescence emitted in an absorption/energy-transfer/ emission (AETE) process (see figure). The configuration of occupied and unoccupied coordination sites of the Tb3+/crown-ether complex is such that only one DPA molecule can bind to it and the complex contains no coordinated water. Relative to the prior technique, the elimination of coordinated water multiplies the sensitivity of detection by a factor of about 10, and the reduction of background luminescence multiplies the sensitivity by another factor of 100, yielding overall improvement of a factor of about 103.

This work was done by Adrian Ponce and Kasthuri Venkateswaran of Caltech for NASA’s Jet Propulsion Laboratory.

In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to

Intellectual Property group JPL Mail Stop 202-233 4800 Oak Grove Drive Pasadena, CA 91109 (818) 354-2240

Refer to NPO-21240, volume and number of this NASA Tech Briefs issue, and the page number.


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Improved Technique for Detecting Endospores Via Luminescence

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This article first appeared in the July, 2002 issue of NASA Tech Briefs Magazine.

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