WO2005001433A1 - Utilisation de la gomme d'acacia pour localiser et conserver un biorisque - Google Patents

Utilisation de la gomme d'acacia pour localiser et conserver un biorisque Download PDF

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Publication number
WO2005001433A1
WO2005001433A1 PCT/US2004/017872 US2004017872W WO2005001433A1 WO 2005001433 A1 WO2005001433 A1 WO 2005001433A1 US 2004017872 W US2004017872 W US 2004017872W WO 2005001433 A1 WO2005001433 A1 WO 2005001433A1
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WIPO (PCT)
Prior art keywords
biological
acacia gum
suspension
biohazard
aqueous solution
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Application number
PCT/US2004/017872
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English (en)
Inventor
Vitaly J. Vodyanoy
Tatiana I. Samoylova
Timothy E. Moore
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Auburn University
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Publication of WO2005001433A1 publication Critical patent/WO2005001433A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N2001/002—Devices for supplying or distributing samples to an analysing apparatus
    • G01N2001/005—Packages for mailing or similar transport of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)

Definitions

  • the present invention relates generally to the containment of biological contaminants and, more particularly, to a method of using a solution of Acacia Gum to contain a biohazard or biological specimen and preserve it in a dormant state for later analysis.
  • Biosensors are used in the health and environmental sciences for rapid detection of specific substances. Biosensors are currently used to detect the presence of pesticides, herbicides, and other compounds; to detect the presence of organic compounds such as alcohols, ammonia, and metals; and, to detect the presence of specific bacteria including algae, fungi, and pathogenic organisms such as Escherichia coli (E. coli) and Salmonella.
  • biosensors include sensing pollution and microbial contamination of air and water, clinical diagnosis of medical conditions, fermentation analysis and control, monitoring and analysis of industrial gases and liquids, monitoring of mining conditions and sensing toxic gases.
  • Biosensors often have a very short shelf life because the antibody or other biological receptor degrades rapidly when exposed to the environment. Like other biological samples, biological receptors need isolation and protection from the environment until ready for use. In field applications, especially, a variety of biological receptors may be needed at any time, depending upon the conditions. There is an unsatisfied need in the art for biological samples that can be protected and preserved without altering or destroying the biological tissue. The demand for safe transport and prolonged storage of biological samples today requires preservation techniques that maintain the integrity and quality of the biological sample.
  • Sensitive biological receptors used in biosensors need to be isolated from the environment, without damaging the receptor, until ready for use. None of the specimen preparation techniques in the art currently meet these needs. There is also a need in the art for biological samples that can be restored to their isolated or prepared state after immobilization, with minimal damage, for later study or use. The current techniques of dehydration and immobilization are irreversible and destroy sample viability. Restoration is particularly critical for the biological receptors in biosensors, which are especially sensitive. There is a need, therefore, for a preservation technique that is both harmless and reversible.
  • the present invention provides a method of using Acacia Gum to contain, reversible preserve, and remove a biohazard from an area.
  • the invention provides a method of containing a biohazard within an area, comprising the steps of preparing a mixture of Acacia Gum in water to form an aqueous solution, and applying the aqueous solution to the area, such that a suspension containing the biohazard is formed.
  • the method further comprises curing the suspension in ambient conditions to form a solid.
  • the step of curing further comprises agitating the suspension.
  • the invention provides a method of removing a biohazard from an area, comprising the steps of preparing a mixture of . to form an aqueous solution, applying the aqueous solution to the area, such that a suspension containing the biohazard is formed, curing the suspension in ambient conditions to form a solid, and removing the solid from the area.
  • the step of curing further comprises agitating the suspension.
  • the invention provides a method of reversibly preserving a biohazard, comprising the steps of preparing a mixture of Acacia Gum in water to form an aqueous solution, applying the aqueous solution to the area, such that a suspension containing the biohazard is formed, curing the suspension in ambient conditions to form a solid, irrigating the solid with an effective amount of aqueous solution to restore the suspension, and separating the suspension such that the biohazard is substantially released from the suspension.
  • the step of curing further comprises agitating the suspension.
  • the aqueous solution comprises a quantity of distilled water, a buffer, and a quantity of one or more compounds selected from the group consisting of potassium chloride, sodium chloride, magnesium chloride, and calcium chloride.
  • the buffer comprises a quantity of 3-(N-morpholino) propanesulfonic acid.
  • Fig. 1 is a photograph of Acacia Gum in powder form, liquid solution, and solid form.
  • Fig. 2 is a series of photographs of bacteria at various stages of immobilization and restoration, according to an embodiment of the present invention.
  • Fig. 3 is a photograph of crystal biosensors coated with a film of Acacia Gum solution, according to an embodiment of the present invention.
  • Fig. 4 is a series of graphs representing the results of experimentation conducted according to an embodiment of the present invention.
  • DETAILED DESCRIPTION The present invention, generally described, provides compositions and methods for the preservation of biological samples.
  • the compositions comprise Acacia Gum, including derivations and modifications thereof which are useful as a reversible preservation solution.
  • Acacia Gum is a complex and highly branched carbohydrate polymer.
  • the central core or nucleus is D-galactose and D- glucuronic acid, to which are attached sugars such as L-arabinose, L-rhamnose, and the like.
  • Acacia Gum is available as thin flakes, powder, granules, or angular fragments which are completely soluble in hot and cold water.
  • Acacia Gum is a natural exudate or sap obtained from any of several plants belonging to the genus Acacia.
  • Acacia Senegal and Acacia Seyal trees are the most commercially exploited species.
  • Acacia Gum typically refers to the gum harvested from Acacia Senegal trees.
  • Acacia plants are leguminous shrubs and trees that grow in warm regions, such as the Republic of the Sudan and the Upper Nile region of eastern Africa, where most of the world's Acacia Gum is harvested.
  • Acacia Gum was widely used in ancient Egypt in the preparation of inks and dyes and is thought to have been used as an adhesive for mummification bindings.
  • An article of commerce for centuries, the name "Arabic Gum” is believed to have been derived from the fact that Acacia gum was typically shipped from Arabian ports to Europe.
  • Acacia Gum is used in the manufacture of printing inks, textile dyes, adhesives, pharmaceuticals, vitamins, confections, foods, beverages, cosmetics, and many other products.
  • Acacia Gum is used to make the water-soluble glue on postage stamps and envelopes, added to candies to prevent crystallization, used as a coating to flavor particles and beverages, added to beer to stabilize the foam, used as an emulsifier of fats in foods, lotions, and soaps, and is the most important gum in the manufacture of ink.
  • the botanical name for the Acacia Gum referred to in this application is Acacia Nilotica (Linn.), N.O. Leguminosae.
  • Acacia Gum is water-soluble, edible, non-toxic, highly uniform, pale in color, and has excellent emulsifying and f ⁇ lm- forming qualities.
  • Acacia Gum consists mainly of high-molecular weight polysaccharides and their calcium, magnesium and potassium salts.
  • Acacia Gum is harvested by tapping the trunk of an Acacia Senegal tree, which causes the gum to seep out and solidify into colorless or pale yellow tear- shaped nodules.
  • the dried nodules are typically gathered by commercially available in the form of white or yellowish flakes, granules, or powder.
  • Acacia Gum powder is plentiful and readily available commercially, at a low cost. When the powder form is dissolved in water, the resulting solution becomes increasingly viscous as the water evaporates, becoming a solid at room temperature. The photograph in Fig.
  • compositions of the invention are useful for the preservation of any biological sample of interest.
  • samples include, without limitation, microorganisms, viruses, bacteria (such as E. coli, Salmonella, Listeria,
  • Animal cells and extracts include, without limitation, semen, sperm, ova, blood, tissue samples, cell samples, urine, saliva, lymphatic fluid, skin, hair, bones, and bone marrow. Additionally, biological samples include proteins, enzymes, antibodies, monoclonal antibodies and the like.
  • biological specimen in an isolated condition indicates a biological sample that has been isolated and substantially purified; meaning that it is substantially or essentially free from components that normally accompany or interact with the sample as found in its natural environment.
  • Acacia Gum powder is readily soluble in water. The solution becomes increasingly viscous as some of the water evaporates.
  • An aqueous Acacia Gum solution is characterized by its reversibility. If more water is added, the viscosity decreases. Even if the solution is permitted to harden or cure into a solid, the addition of water will return the solid to an aqueous solution. Reversibility in this context also refers to the fact that the Acacia Gum solution can be separated nearly completely from the biological specimen after the preservation method of the present invention has been performed.
  • a biological specimen is preserved by being immersed in or otherwise combined with an effective amount of Acacia Gum or an Acacia Gum solution.
  • the amount of Acacia Gum solution will vary depending upon sample size.
  • biological material such as bacteria remain active and motile.
  • the suspension may be stirred to ensure a good distribution of specimen or to speed the evaporation of water and thus accelerate the curing process. Curing may take place in ambient conditions; in other words, at room temperature and at normal atmospheric pressures. When the solution solidifies, the bacteria shrink to about one-half to one-third of their original size.
  • the invention is not bound by any particular mechanism of action, it is postulated that the Acacia Gum solution penetrates the cell membrane of the biological material, possibly replacing the water and resulting in the overall shrinkage observed. Inside the resulting solid, the bacteria remain dormant and may be kept at room temperature.
  • the solid material containing the biological specimen may be made into a powder, pellets, tablets, flakes, plates, capsules, or other forms or containers.
  • the solid is transparent to visible light, a feature that makes it suitable for viewing and for certain optical applications.
  • the solid is water-soluble, the solid is resistant to almost all organic solvents and most acids. To restore the biological material to its isolated condition, the solid is irrigated with an aqueous solution.
  • the amount of aqueous solution needed to change the solid back into a suspension will vary depending upon the sample size.
  • the phrase "effective amount of aqueous solution” is intended to indicate an amount sufficient to transform the solid into a suspension.
  • the aqueous solution used to irrigate the solid contains distilled water, a buffer, and one or more salt compounds such as potassium chloride, sodium chloride, magnesium chloride, and calcium chloride.
  • the buffer is a substance capable in solution of neutralizing both acids and bases and, thereby, maintaining the original pH of the solution.
  • One such pH buffer in common use is 3-(N-morpholino) propanesulfonic acid (also known as MOPS).
  • Another common pH buffer is called a phosphate buffer.
  • a phosphate buffer in one form, contains anhydrous monosodium phosphate and trisodium phosphate dodecahydrate.
  • a phosphate buffer solution may contain dif monosodium phosphate and trisodium phosphate, depending upon the value of the pH to be maintained.
  • the solid When irrigated, the solid gradually dissolves and the biological specimen is again suspended within an Acacia Gum solution. The viscosity of the suspension decreases as more aqueous solution is added. The biological specimen returns to its normal size, absorbing the water lost or exchanged during the curing process.
  • the suspension of biological material and Acacia Gum solution is reversible because it can be separated.
  • the Acacia Gum solution can be removed using common methods of separating mixtures, leaving the biological specimen in its isolated condition.
  • the separation step restores the biological specimen to its former isolated or prepared condition.
  • the phrase "substantially restored” is intended to describe the nearly complete separation of the Acacia Gum solution from the biological specimen and the nearly complete restoration of viability of the biological specimen.
  • a biohazard generally, is a biological agent or condition that presents a hazard to humans or the environment.
  • the release of a biohazard into the environment may occur by accident or, in the case of biological weapons, intentionally. Prompt containment of a biohazard is crucial to reducing its impact on humans and the environment.
  • Environmental conditions generally cause biohazards to disperse and spread.
  • a biological weapon may include an explosion to more rapidly disperse a biohazard over a wide area.
  • the force of an explosive device may force create an aerosol containing the hazardous molecules. Accordingly, the contaminated area or dispersion field may vary in size depending on the circumstances of the release, the forces exerted on the biological agent during release, and the duration of time since the release.
  • an aqueous solution of Acacia Gum may be applied to an area of biological contamination to contain the biohazard and stop it from spreading.
  • the viscosity of the solution may be controlled by varying with the amount of water in the mixture so that it may be spread by any technique, including manually, through hoses, using portable sprayers on foot, by crane, or by air.
  • the application of Acacia Gum solution in one embodiment, forms a suspension containing the hazardous biological agent. In one aspect, the application may require stirring or agitating the suspension to promote the retention of the hazardous biological agent within the solution during the curing process.
  • the solution is permitted to harden or cure into a solid film, by simply allowing the water to evaporate.
  • the hazardous biological agent covered by the mixture is trapped inside the solid film of Acacia Gum and preserved. Once dry, the solid film can be removed from the environment.
  • the preserving quality of an aqueous Acacia Gum solution is particularly well-suited to this application because there is often a need to preserve a biological agent for later study, to discover its exact composition and to discover clues to its origin.
  • the biological agent may be released easily in a secure setting by simply adding water. Also, even though the solid is water-soluble, the solid is resistant to almost all organic solvents and most acids.
  • Biosensors The methods of the invention find particular use in preserving biological samples on biosensors.
  • a biosensor as shown in Fig. 3, is comprised of a biological receptor, an interface, and a signal transducer.
  • the biochemical signal produced when a sample is placed on the biological receptor is converted or translated by the signal transducer into a quantifiable electrical signal.
  • the biological receptor is selected to sense a specific target compound called the analyte.
  • a copper receptor will absorb copper molecules from a sample.
  • the signal transducer converts the activity on the receptor (e.g., the accumulation of copper molecules) into an electrical signal.
  • the signal transducer can detect the increased mass of the biosensor by sensing changes in certain electrical properties.
  • the types of biological receptors in use include, without limitation, enzymes, antibodies, phages, and lipid layers.
  • the biological receptor must be prepared such that it will respond to the analyte. Preparation of the biological receptor includes depositing the biological material onto the of the interface to receive the biological receptor may include chemical etching of the interface, the application of thin membranes, coating the interface with a thin layer of a particular biochemical to serve as an anchor for the biological receptor, or any other of a variety of preparation methods.
  • the phrase, "biological specimen in a prepared condition," as used herein indicates a biological receptor that has been isolated and deposited upon the biosensor interface using any preparation technique that renders the receptor ready for its intended use.
  • the signal transducer is typically an electrode connected to the interface to measure any change in the receptor when the sample is introduced.
  • Signal transducer systems include, without limitation, piezoelectric crystals, conductimeters, enzyme-sensing electrodes, thermistors, optoelectronic and fiberoptic devices, field-effect transistors, gas-sensing electrodes, and ion-selective electrodes.
  • the signal transducer itself may be a pH-electrode, an oxygen electrode, or a piezoelectric crystal.
  • the biological receptor is deposited in a film onto a piezoelectric crystal, which serves as the interface. An electrode attached to the crystal acts as the signal transducer.
  • the quartz crystal is oscillated at a known frequency based on its total mass, including the mass of the film receptor.
  • the total mass will change when the antibodies in the receptor bind to the analyte.
  • the frequency of the crystal oscillation will change, and the change in frequency is measured by the signal transducer. Because frequency and mass are related, the additional mass can be calculated, indicating the precise amount of the analyte present in the sample.
  • the Biosensor Experiment A biosensor with a biological receptor comprised of Salmonella bacteria was covered with a film of Acacia Gum solution. After curing and storage at room temperature for a period of four (4) days, the bacteria were released by irrigation with water containing 55.0 milli-Molar potassium chloride, 4.0 milli-Molar sodium chloride, 1.0 milli-Molar magnesium chloride, 0.1 milli-Molar calcium chloride, and 2.0 milli-Molar 3-(N-morpholino) propanesulfonic acid, used as a pH buffer. Preliminary data was obtained demonstrating the sensitivity of the restored sensors compared to the uncoated sensors, as shown in Fig. 4 and Table One.
  • the biosensors used in this experiment were the PM-700 series quartz sensor crystals available from Maxtek, Inc.
  • the output of the sensor crystal corresponds to the change in total mass.
  • the signal transducer measures the change in the crystal in millivolts (mV).
  • mV per decade refers to the voltage change for each order of magnitude change in the bacterial concentration.
  • the bacterial suspension of approximately 10 9 cells per milliliter was diluted 10, 100, and 1000 times, respectively.
  • the relative concentrations of bacteria were, therefore, 1, 10 "1 , 10 "2 , and 10 "3 . Accordingly, the logarithms (shown in Fig.
  • the methods of the invention are useful in preserving animal cells and extracts, such as sperm.
  • the isolation and preservation technique of the present invention was used to temporarily and reversible preserve bull sperm. A sample of bull sperm was immobilized in Acacia Gum solution, where it remained at room temperature for a period of four (4) days before being released by irrigation with water. Although reproduction was not tested, the bull sperm showed no difference in motility when compared to the initial sample.
  • the present invention may be used to preserve bull sperm for transport or storage, at room temperature, without significant damage to the sperm.
  • the cryogenic preparation and storage of bull sperm is expensive and destructive because of crystalline structures formed during freezing.
  • the present invention does not introduce crystals or other destructive structures into the sample and it is much less expensive.
  • Bacterial Cultures The methods of the present invention are also useful in preserving samples of bacteria. Two separate experiments were conducted to test the response and subsequent viability of bacteria suspended within an Acacia Gum solution. In a first experiment, separate samples of Escherichia coli 0157 (E. coli) bacteria and Salmonella bacteria were immobilized in Acacia Gum solution, where each sample remained at room temperature for a period of seven (7) days. The bacteria were released by irrigation with water containing a phosphate buffer (pH 7.4) containing 2.7 milli-Molar potassium chloride and 137 milli-Molar sodium chloride. The released bacteria showed no difference in motility when compared to the initial culture. The bacteria reproduced normally. Fig. 2 shows the Salmonella bacteria at different stages of the experiment.
  • E. coli Escherichia coli 0157
  • Salmonella bacteria were immobilized in Acacia Gum solution, where each sample remained at room temperature for a period of seven (7) days.
  • the bacteria were released by irrigation with water containing a
  • Slide a shows the bacteria immersed in the Acacia Gum solution.
  • Slide b shows the bacteria immobilized within the Acacia Gum solution, which has become a solid at room temperature. Notice that the bacteria in Slide b are somewhat smaller. After remaining immobilized for seven (7) days, the bacteria were irrigated with an aqueous solution. The restoration process is shown i Slide c shows the condition of the bacteria after one minute. Some motion was observed after two minutes, shown in Slide d.
  • Slide e shows the condition of the bacteria after three minutes. After ten minutes, as shown in Slide f, the bacteria have returned to their normal size, absorbing the water lost during the immobilization or curing process.
  • two additional samples of E. coli and Salmonella bacteria were immobilized in Acacia Gum solution for a period of twenty-one (21) days, with the same results. The bacteria showed no difference in motility when compared to the initial culture and the bacteria reproduced normally.
  • the present invention offers a method of reversibly preserving biological specimens in a variety of contexts.
  • the isolation and preservation techniques of the present invention could be used, without limitation, for isolating microbial cultures for storage or shipment, blood isolation and storage, biohazard isolation and storage, time-release capsules for pharmaceuticals, biodegradable packaging, soluble prostheses and implants, surgery, and forensics.
  • the Acacia Gum solution and the isolation and preservation techniques of the present invention represent a simple, rapid, and inexpensive alternative to many of the biological preservation techniques in use today.
  • Acacia Gum is organic, water-soluble, bio-compatible, biodegradable, and non-toxic.
  • the preservation of biological specimens with Acacia Gum is reversible and causes little or no damage to the specimen. While this invention has been described in specific detail with reference to the disclosed embodiments, it will be understood that many variations and modifications may be effected without departing from the invention as described in the appended claims.

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Abstract

L'invention porte sur des compositions et sur des procédés de localisation et de conservation réversibles d'échantillons biologiques. Les compositions comprennent de la gomme d'Acacia, y compris des dérivations et modifications de celle-ci qui sont utilisées sous forme d'une solution de conservation réversible. L'invention porte sur un procédé d'utilisation de la gomme d'Acacia pour isoler et conserver de manière réversible un échantillon biologique à l'état dormant à température ambiante sur une durée prolongée, avec un minimum de dommage pour l'échantillon. Les compositions et les procédés décrits peuvent être utilisés pour créer des échantillons biologiques conservés de manière réversible et des récepteurs biologiques destinés à être utilisés dans des biocapteurs. Les compositions et les procédés décrits peuvent aussi être utilisés pour localiser un biorisque et le conserver pour une analyse ultérieure.
PCT/US2004/017872 2003-06-13 2004-06-07 Utilisation de la gomme d'acacia pour localiser et conserver un biorisque WO2005001433A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971783A (en) * 1982-11-22 1990-11-20 The University Of Va Alumni Patents Foundation Tissue processing for immunofluorescence microscopy
WO2002043779A2 (fr) * 2000-12-01 2002-06-06 Auburn University Utilisation de la gomme d'acacia pour isoler et conserver une substance biologique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971783A (en) * 1982-11-22 1990-11-20 The University Of Va Alumni Patents Foundation Tissue processing for immunofluorescence microscopy
WO2002043779A2 (fr) * 2000-12-01 2002-06-06 Auburn University Utilisation de la gomme d'acacia pour isoler et conserver une substance biologique
US20030138939A1 (en) * 2000-12-01 2003-07-24 Vodyanoy Vitaly J. Use of acacia gum to isolate and preserve biological material

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