A method of detecting bacterial spores incorporates
- A method of lateral-flow immunoassay in combination with
- A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound.
The second-mentioned component method, denoted the method of DPA-triggered Tb luminescence, was described in somewhat more detail as a precursor of a related development reported in "Improved Technique for Detecting Endospores via Luminescence" (NPO-21240)NASA Tech Briefs, Vol. 26, No. 7 (July 2002), page 56.
The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both
- High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and
- Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method. The sensitivity afforded by the present method is so much greater that whereas the previously reported detection limit of lateral-flow immunoassay was 105 spores/mL, the estimated detection limit of the present method is 100 spores/mL.
DPA in a 1:1 complex with Ca2+ ions is present in high concentration in bacterial spores, and has not been observed in any lifeforms other than bacterial spores. Hence, DPA is an indicator molecule for the presence of bacterial spores. Fortuitously, DPA is also a classic inorganic-chemistry ligand that binds metal ions with high affinity. When bound to Tb3+ ions, DPA triggers intense green luminescence of the ions under ultraviolet excitation. The intensity of the luminescence can be correlated with the number density of bacterial spores per milliliter. Moreover, the concentrations of compounds that could potentially give rise to spurious luminescence are typically much smaller than the concentration of DPA, and the strengths with which they bind to Tb3+ are of the order of a millionth of that of DPA, so that the desired luminescence signal appears against a dark background.
The figure summarizes the steps of the present method. A sample suspected of containing bacterial spores is prepared by suspending raw sample material in an aqueous solution that contains Tb3+ ions. A volume of ≈100 -L of the sample is placed on a test strip - a nitrocellulose membrane on which species-specific antibodies are bound in an area denoted the sample region (area A in the figure). Capillary action moves the spores along the strips. In the sample region, specific binding of membrane-bound antibodies captures and immobilizes the bacterial spores. Next, the strip is exposed to microwave power to release DPA from the spores. The released DPA binds to the Tb3+ ions in the solution. Hence, when the strip is exposed to ultraviolet light, the Tb3+ ions luminesce green, signaling the presence of the bacterial spores from which the DPA was released.
At the same time, a volume of ≈100 -L of a similar solution containing a known concentration of Bacillus subtilis, to be used as a standard, is placed on a similarly prepared, parallel membrane denoted the standard strip, which includes an antibody-coated area designated the standard region (area B in the figure). The standard strip is subjected to the same process as is the test strip. A combination of green luminescence from the region B and a change in color in regions I of both strips indicates that the assay has worked properly. In that case, the ratio of between the intensity of luminescence in region A and that in region B is proportional to the number density of bacterial spores in the sample. The entire assay can be performed in 10 minutes or less.
This work was done by Adrian Ponce 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
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Refer to NPO-30469.