Chemistry: analytical and immunological testing – Optical result – Including reagent preparation
Reexamination Certificate
2003-02-04
2004-02-10
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
Including reagent preparation
C436S164000, C436S095000, C436S071000
Reexamination Certificate
active
06689620
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the use of giant unilamellar vesicles (GUV) in the colorimetric detection of chemical and biological agents by simple receptor-ligand interaction.
BACKGROUND OF THE INVENTION & DESCRIPTION OF RELATED ART
Diacetylenes and their analogues upon polymerization produce a deep blue colored polydiacetylene polymer. The presence of conjugated single, double, and triple bonds in polydiacetylene backbone is responsible for the deep color. See B. Tieke, G. Lieser, J. Colloid Interface Sci., 88, 471(1982).
A blue to red color shift in polymer-backbone is observed when the polymer goes through mechanical or thermal stress. See A. Singh, R. B. Thompson, J. M. Schnur, J. Am. Chem. Soc. 108, 2785(1986). This stress associated color change in polydiacetylenic backbone has been used in the development of various detector and sensor schemes.
This idea has been extended to the detection of molecular species which cause stress on the polymer backbone upon binding to the molecular sites covalently attached to the polydiacetylene backbone. For example, polydiacetylene films containing glycolipid sites have been used in the detection of biological species such as cholera toxin. See Charych et al., Science, 261, p. 585-588 (Jul. 30 1993).
The conventional approach of thin polydiacetylenic film faces the difficulty of making the thin film and then transferring it onto a substrate. Reading the film spectrophotometrically poses an additional problem. The film is generally deposited on the glass slide substrate and the binding experiment is done in the solution for about 20 minutes. The color changes are read spectrophotometrically, a cumbersome process which may lead to some inaccuracies due to changes in the medium during the course of the experiment.
Vesicles serve as the best substrates because of their large surface area, good dispersion behavior, and availability of surface available receptor sites for toxins. But the diacetylene moiety in a small vesicle does not polymerize efficiently due to its small radius of curvature. Therefore color changes due to stress on the polymer backbone, transferred by ligand binding, may not be visible.
Multilamellar vesicles (MLVs) may polymerize better than small unilamellar vesicles (SUVs), but they will partially transform into other structures such as tubules in most cases when cooled below their phase transition temperature. See P. Yager, P. E. Schoen, Mol. Cryst. Liq. Cryst., 106, 371(1984). This cooling step is needed to permit topotactic polymerization of diacetylene. Topotactic polymerization relates to ordering the neighboring polymerizable diacetylenic functionalities in a parallel to each other fashion. This ordering is acquired in diacetylene containing chains by cooling the lipid vesicles below their phase transition temperature or T
m
. See Ticke, B., et al, J. Polym. Sci., Polym. Chem. Ed. 7, 1631-1644 (1979) and Lever et al, Biochim. Biophys. Acta 732, 210-218 (1983).
The diacetylenic SUVs do remain stable at temperatures down to about 2.4° C., almost 40 degrees below T
m
, where differential scanning calorimetry shows that a phase transformation occurs. See T. G. Burke, et al., Chem. Phys. Lipids., 48, 215 (1988). Although these small vesicles do not turn into tubules upon cooling, as discussed above, these SUV diacetylenes fail to polymerize to provide an extended conjugation due to curvature constraints. Such extended conjugation is desired to provide darker color which results in better visibility.
Giant unilamellar vesicles (GUVs) produced from diacetylenic phospholipid, 1,2 bis (heptacosa-8,10-diynoyl)-sn-glycero-3-phosphocholine (DC
6,15
PC) have recently been prepared which are generally 10 to 100 times larger than typical vesicles by applying an electric field to the aqueous dispersion maintained above its chain melting transition temperature (T
m
) of 58.9° C. See Alok Singh, Paul E. Schoen, Marie-Alice Guedeau-Boudeville, Chem. Phys. Lipids., 94, 53-61 (1998). There has been no disclosure of these new GUVs being used in colorimetric sensors.
OBJECTS OF THE INVENTION
It is an object of this invention to produce giant unilamellar vesicles (GUVs) with a large radius of curvature which can polymerize effectively.
It is a further object of this invention to produce giant unilamellar vesicles with a large radius of curvature which are polymerized and which have a polymer membrane that allows the free transport of contents across the membrane bilayer so as to eliminate the risk of any color change due to osmotic shock.
It is a further object of this invention to produce giant unilamellar vesicles with a large radius of curvature which are polymerized and large in size so they may be handled and manipulated individually.
It is a further object of this invention to produce giant unilamellar vesicles with a large radius of curvature which have a size of at least 50 microns.
It is a further object of this invention to produce a colorimetric detector of chemical and biological agents utilizing giant unilamellar vesicles with a large radius of curvature so as to make customized arrays to enhance detection.
It is a further object of this invention to produce a colorimetric detector of chemical and biological agents utilizing giant unilamellar vesicles with a large radius of curvature so that individual giant unilamellar vesicles may be probed for color change due to site specific binding.
These and further objects of the invention will become apparent as the description of the invention proceeds.
SUMMARY OF THE INVENTION
A colorimetric detector for chemical and biological agents or toxins is made of a giant unilamellar vesicle (GUV) having a membrane bilayer which is polymerized to stabilize the giant unilamellar vesicle and to provide extended conjugated polymer backbone, and there is at least one incorporated molecular recognition site for the chemical and biological agents or toxins. The GUV is preferably a polymerizable diacetylenic GUV. In this case, the polymerization to stabilize the giant unilamellar vesicle is done by crosslinking the acyl chains in the membrane bilayer of the diacetylenic GUV. Upon the binding by the chemical and biological agents or toxins, the detector exhibits a color change in the visible region of the spectrum. Examples of incorporated molecular recognition sites are di or polysachharides substituted with a long alkyl chain preferably containing diacetylenic moiety. Preferable examples are N-octadecyl maltobionamide (C-18 maltonamide) and N-octadecyl lactobionamide (C-18 lactonamide). The GUV is preferably made from 1,2 bis-(alkadiynoyl)-sn-glycero-3-phosphocholine DC
m,n
PC where m=2-16 and n=7-16, and m+n≦20 carbon atoms. A particularly preferred material is 1,2 bis-(Tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine DC
8,9
PC. The GUV in the detector has a large radius of curvature of at least 50 microns and the size of the GUV is about 10-300 microns. The GUV can be used on a substrate to detect the presence of specific species or it can be used by itself as a substrate to detect the presence of specific species.
The colorimetric detector is used for the detection of chemical and biological agents or toxins in a test sample by exposing the test sample to the colorimetric detector which is made of giant unilamellar vesicles (GUV) having a membrane bilayer which is polymerized to stabilize the giant unilamellar vesicle and to provide extended conjugated polymer backbone and which has at least one incorporated molecular recognition receptor site for the chemical and biological agents or toxins. As a result of the interaction of the chemical and biological agents with the receptor sites a colorimetric detection signal is produced.
The colorimetric detector can be used in a colorimetric detector apparatus for detecting chemical and biological agents or toxins. The apparatus has a chamber with a series of passageways for analyzing the sampling fluid containing the colorimetric detector described above. There is a chamber inlet for sampling a fluid cont
Schoen Paul
Singh Alok
Forman Rebecca
Gakh Yelena
Karasek John J.
The United States of America as represented by the Secretary of
Warden Jill
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