Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Calorimeter
Reexamination Certificate
2001-07-10
2003-11-25
Chin, Christopher L. (Department: 1641)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Calorimeter
C422S105000, C422S105000, C422S105000, C422S105000, C435S808000, C435S283100, C435S288700, C436S165000, C436S175000, C600S562000, C600S566000
Reexamination Certificate
active
06652807
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to apparatus for collecting and concentrating analytes within a liquid medium for the purpose of identifying and quantifying the analytes using Raman, surface enhanced Raman scattering (SERS), infra-red (IR) or fluorescence measurement techniques.
Ciguatoxin is the major causative toxin in ciguatera fish poisoning, a disease which remains a serious fisheries and public health problem worldwide wherever reef fish are caught and consumed. Annual worldwide estimates of people afflicted by ciguatera poisoning range from 500,000 to 1,000,000. In the United States, ciguatera is the single, leading cause of seafood poisoning. Worldwide, only 10 percent of all ciguatera cases are probably reported (Lewis, 1986). During the past five years, increased reports of ciguatera have also been documented in Mexico (Lechuga-Deveze and Sierra-Beltran, 1995; Arcila-Herrera et al., 1998; Sierra-Beltran et al., 1998), California (Barton et al., 1995), and temperate countries such as Canada (Bruneau et al., 1997; De Haro et al., 1997; Kelmme and Losch, 1997; Sanner et al., 1997; Blume et al., 1999), that import fish from ciguateric regions or whose residents travel to endemic ciguatera areas and contract the disease.
Although the ciguatera toxin-producing organism may not be considered a traditional pathogen, it does have significant global impact on human health, fisheries and their dependent economies. Ciguatera often manifests itself similar to a severe flu, causing weakness, diarrhea, muscle pain, joint aches, nausea, chills, headache, sweating and dizziness. These symptoms are often accompanied by numbness or tingling around the mouth and in the extremities, and a strange sense of temperature reversal where hot items feel cold to the touch and cold objects feel hot. Symptoms typically persist for days or weeks, but may last for months or years. Deaths are rare but can occur in severe or complicated cases.
The occurrence of ciguatera toxins in many fishes can prevent many commercially important fishes from being utilized in states or island nations with limited resources. Consequently, ciguatera can have devastating impacts on the development of small-scale commercial fisheries. In 1984, the economic losses in Florida, the Caribbean and Hawaii due to ciguatera totaled over $10 million annually. Given the rise of inflation and the continued existence of ciguatera, this figure today represents a significant loss in revenue for the fishing industries in these areas as well as those in other ciguatera affected countries. Thus, the areas that need a test method to rapidly screen fish for ciguatoxin include ciguatera endemic areas such as Hawaii, Florida, Guam, the Philippines, Japan, and the Caribbean; commercial fisheries in these areas as well as countries importing seafood from these areas; and diagnostic laboratories.
The major marine toxins associated with ciguatera poisoning have been attributed to the class of chemicals designated ciguatoxin (CTX) and its congeners. Currently, Oceanit Test Systems (OTS), a subsidiary of Oceanit Laboratories, Inc. (Oceanit), offers the only commercially available CTX detection kit, Cigua-Check®, developed by Oceanit and marketed since October 1997. Although past research has proven that CTX screening using the monoclonal antibody used in the Cigua-Check® system is effective in preventing ciguatera and can provide results within one hour, this method is designed primarily for small-scale home or field use. In order to screen potentially toxic fish from ciguatera-endemic areas, however, an even simpler, larger scale technique needs to be designed to prevent consumption of seafood tainted with CTX as well as to aid in confirming ciguatera cases caused by this toxin in the United States as well as other affected nations.
Measuring trace amounts (in parts per trillion or less) of analytes usually requires specialized techniques. Recent advances in Raman, IR and fluorescence spectroscopy have enabled increased sensitivity to detect such analytes. While methods based on identifying analytes based on their unique chemical and physical properties exist, most require considerable sample preparation and the use of expensive detectors. The need exists for techniques to measure trace amounts of analytes simply and rapidly. One example of such a trace analyte is CTX.
There is a need to prevent human illness due to ciguatera toxins by creating an innovative method to detect these harmful toxins in fish before they are incidentally ingested.
SUMMARY OF THE INVENTION
The invention measures ciguatoxin more easily and rapidly than current technologies and thus fulfills the need for a sensitive and effective method for ciguatoxin detection suitable for large-scale screening of potentially toxic fish.
The ciguatoxin molecule itself fluoresces when exposed to incident light of certain wavelengths. Why does a long hydrocarbon chain molecule such as ciguatoxin fluoresce? In Quantum Mechanics, the “Particle in a Box” scenario describes what it takes to get electronic transitions in the visible spectrum. Large organic molecules exhibit similar physics to the particle in a box. For a small molecule such as hydrogen, the transition from the ground state to the first excited electronic state occurs far in the ultraviolet. In order for the first excited state to be at a lower energy the width of the “box” or length of the molecule in this case would have to be increased. Lower energetic transitions correspond to longer wavelengths, which are observed in the fluorescence process.
The multiple side groups on long hydrocarbon chains create a de-localized electron cloud over the molecule, which have transitions that are low enough in energy to lie in the visible between the ground level and the first excited state. Isolated molecules have isolated energy levels, and larger molecules have a dense set of energy levels. For each electronic state there is a whole set of vibrational states and for each vibrational state there is a whole set of rotational states.
In a liquid, the molecules are free to move around, but for larger molecules, as soon as they move by even a fraction of their diameter they collide with a neighboring molecule. These collisions will cause decay from excited states to lower excited states or to the ground state. This collisional decay process broadens the absorption spectrum substantially. The isolated discrete electronic, vibrational, rotation levels broaden and overlap into what looks like a continuum of levels resulting in a band of energy levels.
In thermal equilibrium the population of electrons in the energy levels follow the Maxwell Boltzmann distribution. The range of energies is so broad that the molecules are all near the bottom of the ground state within each energy band. Transitions occur from anywhere in the band of ground levels to anywhere in the first excited state.
An example of this phenomenon can be seen with rhodamine
6
G “Texas Red,” a reddish-orange colored dye used in dye lasers and as a “fluorescent beacon” in laser scanning confocal microscopy. Single molecules of rhodamine
6
G have also been detected using SERS. If a molecule such as this absorbs energy it is raised into the first excited state, and will be in a high lying vibration rotation state well above the bottom of the first excited state. This excess energy is lost very rapidly due to collisions with the neighboring molecules in the liquid, lowering the energy to the bottom of the band of first excited states. Optical transitions can occur from the bottom of the first excited state to anywhere in the band of ground states. This mechanism establishes a natural population inversion, and the sample of dye will have strong fluorescence when illuminated with light. This occurrence may also explain why the ciguatoxin molecule fluoresces.
The invention has many advantages over present ciguatoxin detection techniques. First, it requires far less time to analyze unknown samples, in the range of minutes as opposed to hours. Second, because th
Ebesu Joanne S. M.
Pernambuco-Wise Paul
Chin Christopher L.
Counts Gary W.
Narasimhan Mara P.
Oceanit Test Systems, Inc.
Way James Creighton
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