WO1999042613A1 - Methods and additives for microtagging fluids - Google Patents

Abstract

Methods and additives for tagging and subsequently detecting commercial products, including industrial liquids, are disclosed. These methods permit the identification of such tags after very high, say 10-11 dilutions. The present invention encompasses a method of microtagging a liquid, subsequently detecting that the liquid has been microtagged and identifying the liquid, which comprises (i) adding to the liquid an additive comprising at least one signal means which may be detected after extreme dilution, and a coding means to aid in identification of the liquid; (ii) obtaining a sample of the liquid contaning said additive; (iii) detecting the presence in the liquid sample of said at least one signal means; (iv) and decoding said coding means, thereby detecting that the liquid had been microtagged and identifying the liquid sample.

Description

METHODS AND ADDITIVES FOR MICROTAGGING FLUIDS

BACKGROUND OF THE INVENTION

This invention relates to the marking of materials for the purpose of later identifying their origin or confirming their integrity. In particular, methods of marking fluids are disclosed, whereby the marker may subsequently be detected after extreme dilution.

There are many circumstances in which it is desirable to unobtrusively mark materials so that their origins can be later be ascertained. Prior art teaches macroscopic tags on trade items which maintain their physical presence and integrity, such as product descriptors on clothing or water marks or magnetic codes on currency. More recentiy, holographic insigmas have been used extensively for product copyright purposes. There are markers for explosives, in which there is a local dispersal of macroscopic tags in the explosion. There are few markers available for more demanding applications, in which the markers constitute minuscule proportions (e.g. less than parts per billion of the host bulk material), and/or in which the host material may become extremely dispersed or diluted by either accident or design. For example, Slater, U.S. 5,643,728 relates to a tagging system using exposed nucleic acids. Such unobtrusive tags may be referred to as "microtags", and methods of unobtrusively marking liquids as "microtagging".

Spills or leaks of petroleum (meaning all petroleum derivatives and more generally hydrocarbons and their chemical derivatives) are unfortunate but all too common. The presence of microtags in a containing vessel would support identification of the spill's source. The vessel might be a ship, barge, truck, railcar, pipeline or tank at a petroleum tank farm. In the case of ship and/or barge spills, major fines are now being assessed on die basis of circumstantial evidence, e.g. that the tanker's path was through the spill location. If the contents of the tanker were microtagged at the start of a voyage, its innocence or culpability could be later established by the respective presence or absence of its microtags in the spill area.

A second example area includes the counterfeiting of well known products. Many brand name luxury products such as perfumes and alcoholic beverages are being counterfeited. Detection of such counterfeits has considerable commercial value. Also, many bulk commercial products, e.g. fuels, are mixtures of plurality of components. Dishonest operators are known to replace more expensive components with cheaper ones, which may lead to inferior performance. For example, for a case of lubricants, the replacement can lead to premature equipment aging, e.g. engine break-down. More generally, a complete audit trail from wellhead to refinery to consumer will be of considerable commercial importance for the petroleum industry.

Third, there are applications where the origin/distribution channels of illegal materials is to be elucidated. Such is the case for chemicals used in the preparation of narcotics, explosives, or toxic materials such as nerve gasses. Fourth, sufficiently lightweight microtags would also be useful in ascertaining the origin of airborne pollutants which should have been contained at a manufacturing or utilization site.

Fifth, there is also a class of applications wherein the time of production and/or distribution of the product is of importance. For pharmaceuticals with specified shelf lives, great illegal profits can be gained by selling the aged items after repacking in falsely dated containers.

For certain agricultural products similar dating might be desirable. There is a clear need to have some type of internal time system independent of a container's claims.

Finally, it would be advantageous to have a way of tagging different liquids involved in the operation and functioning of various machinery. In the study of wear and friction processes, for example, such a method would be valuable in monitoring valve integrity. In complex industrial systems such as petrochemical or pharmaceutical installations, the failure of a valve can lead to major problems. The leak or loss of material in question is one such problem, but more significant is the contamination of other materials in the system by the leaking substance. By tagging different pipes or containers within a system with different microtags which may be distinguished from each other, the problem of determining which valve is leaking is immediately circumvented.

There are several technical challenges to any microtagging system:

• a need for reliable detection of very small quantities, e.g. at dilutions as high as one part per trillion; • a need for high encoding capability to support the scale of commerce;

• a recovery strategy supporting detailed readout of forensic codes;

• an unobtrusiveness that bars counterfeiting of microtags.

• stability and robustness which prevents intentional tampering and accidental tag loss. The invention involves implementations which satisfy all these requirements and are relatively inexpensive. SUMMARY OF THE INVENTION

The present invention encompasses a method of microtagging, or unobtrusively marking, a fluid material, subsequently detecting that the material has been microtagged, and identifying the material. Preferably, the fluid is a liquid although it may be a gas or a fine, flowable solid particulate. In the method of microtagging a liquid, a microtag is an additive which comprises at least one stable signal means which may be detected after extreme dilution, and may serve as a preliminary classification of origin, or as a "code key", which is used to indicate (i) that the liquid of interest has been microtagged, and (ii) the nature of the code itself. The code itself, or coding means, is also a part of the microtag, and may be used to identify a specific liquid. Thus, the present invention encompasses a method of microtagging a liquid, subsequently detecting mat the liquid has been microtagged and identifying the liquid, which comprises (i) adding to the liquid an additive comprising at least one signal means which may be detected after extreme dilution, and a coding means to aid in identification of the liquid; (ii) sampling a portion of the liquid containing said additive; (iii) detecting the presence in the liquid sample of said at least one signal means; (iv) and decoding said coding means, thereby detecting that the liquid had been microtagged and identifying the liquid sample.

The liquid may be of any type or origin, for instance a perfume, a lubricant or a pharmaceutical, but preferably is or comprises a petroleum or a hydrocarbon or a chemical derivative thereof. Depending on the liquid and the stability and structure of a particular signal or coding means, a signal or coding means may be added directly to the liquid, or may first be attached to or enclosed in a particulate. The particulates may be microbeads, microspheres or microscopic envelopes formed from thin films (microcontainers), and may be magnetic to aid in the concentration and isolation of said particulates for decoding of the coding means. Alternatively, the particulate may be a bacterium or a bacterial spore. When a sporulating bacterium is used as a container for the coding means, the coding means may be a nucleic acid or a nucleic acid which encodes a polypeptide. The microtagging method will then involve transforming the bacteria with the nucleic acid microtag, or with a nucleic acid encoding a polypeptide, growing the bacteria until sporulation occurs, and mixing the spores with the liquid to be microtagged. Accordingly, the detection step will then involve recovering the spores, exposing the spores to an appropriate medium such that germination is

3 - induced, growing the germinated bacteria such that the nucleic acid coding means is replicated, and decoding said coding means.

When a nucleic acid is used as the coding means, detection may involve PCR, sequencing or hybridization, or any combination thereof. Because a wide variety of different codes may be used by varying the content of the nucleotide sequence, nucleic acids are particularly preferred as a coding means. However, since a nucleic acid may be simply and quickly detected using hybridization or PCR without determining the actual sequence of nucleotides, it should be understood that a nucleic acid can also serve as a preliminary signal means.

Where the coding means is a polypeptide, the detection step may involve a specific enzymatic reaction or an immunoassay. Because of the virtually endless combinations of specific antigen/antibody matches, polypeptides are also a particularly preferred coding means. It should be understood by those of skill in the art that, depending on the sensitivity of the assay, an immunoassay may also be a relatively quick way to determine the presence of a microtag. Thus, a polypeptide may also be a signal means. Furthermore, depending on the stability of the polypeptide, it may be attached or enclosed in a particulate, expressed in a bacterium, or added directly to the liquid to be microtagged.

The "polypeptide" may be more generally defined here as a first molecule having a binding affinity for a second molecule. Particular examples include an antigen and an antibody; a hormone and a hormone receptor; a poly nucleotide and a complementary poly nucleotide; and biotin and either avidin or streptavidin. These examples are by no means exhaustive, and could include other interactive molecules as would be clear to those of skill in the art. When a binding pair component is used as a microtag, particularly one which has a binding affinity for a second molecule, the binding pair may be used to initially concentrate or isolate the microtag from the marked liquid. When the microtag is added directly to the liquid without being attached to or enclosed in a particulate, it may take the form of any compound which may be detected directly and/or concentrated and isolated. For example, in this case the microtag may be a chemical compound, a radiolabel or a fluorescent label.

Where the microtag is a radiolabel or a radioisotope, it is preferably a multiphoton detection-compatible radioisotope, and more preferably in the family of halogens, i.e. Br⁷⁶(16.5 h), Br⁷⁷(2.6 d); I¹²³(10 h), I¹²⁴(4.2 d), I¹²⁵(60 d), I¹²⁶(13.2 d). Radioactive iodine is especially preferred, but any member of the family of electron capture (EC) emitters is suitable. Positron- gamma (PG) emitters may also be used.

The radioisotopes may also belong to the family of lanthanides, including La¹³⁵ (19.8 h); Ce¹³³(6.3 h), Ce¹³⁴(3.0 d), Ce¹³⁵(22.0 h), Ce¹³⁷( 9.0 h), Ce¹³⁹(140 d); Nd¹⁴⁰( 3.3 d); Pm^{1 3}(265 d), Pm¹⁴⁴ (440 d), Pm¹⁴⁵(18 y), Pm¹⁴⁶ (710 d), Pm^158m(40.6 d); Sm¹⁴⁵(340 d); Eu¹⁴⁵(5.6d),

Eu^146m(1.58d), Eu¹⁴⁶(4.6d), Eu¹⁴⁷(24d), Eιf⁴⁸(54d), Eιr⁴⁹ (120d), Eιf^50m(14h), Ei ⁵⁰ (5y), Etf⁵² (13 y); Gd¹⁴⁶(48 d), Gd¹⁴⁷(35 h), Gd¹⁴⁹(9 d), Gd ¹⁵^(120 d), Gd ¹⁵^200 d); Tb ¹⁵ 1 h), Tb ^15;tl8 h), Tb¹⁵³(2.58 d), Tb^154m(8 h), Tb¹⁵⁴(21 h), Tb¹⁶⁵(5.4 d), Tb¹⁶⁰(73 d); Dy¹⁵⁵(10 h), Dy¹⁵⁷(8.2 h); Tm¹⁶⁵(1.21d), Tm¹⁶⁷(9.6 d), Tm¹⁶⁸(85 d); Yb¹⁶⁹(32 d); Lu¹⁶⁹(1.5 d), Lu¹⁷⁰(2.0 d), Lu¹⁷¹ (8.3 d), Lu¹⁷²(6.7 d), Lu¹⁷³(1.3 y), Lu^174m(165 d); Hf¹⁷³(24 h), Hf¹⁷⁵(70 d); Ta¹⁷⁵(ll h), Ta¹⁷⁶(8.0 h),

Ta¹⁷⁷(2.21 d), Ta¹⁷⁹(1.6 y), Ta^180m(8.1 h).

In addition, the multiphoton emitters may belong to families of heavy metals and actinides, including W¹⁸¹(130 d); Re¹⁸¹(20 h), Re^182m(13 h), Re¹⁸²(64 h), Re¹⁸³ (71 d), Re^184ra(2.2 d), Re¹⁸⁴(50 d), Re¹⁸⁶(90 h); Os^183m(10 h), Os¹⁸³(12 h), Os¹⁸⁵(94 d); Ir¹⁸⁵(15 h), Ir¹⁸⁷(12 h), Ir¹⁸⁸(1.71 d), Ir¹⁸⁹(l l d), Ir¹⁹⁰(l l d), Ir¹⁹²(74 d); Pt¹⁹¹(3.0 d); Au¹⁹³(15.8 h), Au¹⁹⁴(39 h), Au¹⁹⁵ (200 d),

Au¹⁹⁶(5.55 d); Hg^193m(l.l d), Hg¹⁹³(6 h), Hg¹⁹⁴(130 d), Hg^l.όό d), Hg¹⁹⁵(9.5 h), Hg^197m(24 h), Hg¹⁹⁷(2.71 d); Tl^20O(1.08 d), Tl²⁰¹(3.04 d), Tl²⁰²(12 d), Tl²⁰⁴(3.9 y); Pb^20O(21 h), Pb²⁰¹(9.4 h), ?b™(2M d); Bi²⁰³(12.3 h), Bi²⁰⁴(11.6 h), Bi^206m(15.3 d), Bi²⁰⁶(6.3 d), Bi²⁰⁷(30 y); Po²⁰⁶(8.8 d); At²¹⁰(8.3 h), At²¹¹(7.2 h); Rn²¹¹(16 h), Ac ²⁶(29 h); Pa²²⁸(22 h), Pa²²⁹(1.5 d); U²³¹(4.2 d); Np²³⁴(4.4 d); Pu²³⁴(9 h), Pu²³⁷(45.6 d); Am²³⁹(12 h); Cm²⁴¹^ d); Bk²⁴⁵(4.95 d), Bk^l.δ d).

When a chemical compound is used in the microtag, it may be any chemical compound that is distinguishable from the natural environment of the liquid and may be detected as a microtag. Preferably, the chemical compound is soluble in the liquid to be microtagged. Because multiphoton detection drastically increases the sensitivity of microtag detection when multiphoton emitters are used, the microtags of the present invention will preferably contain chemical compounds derivatized to a radioisotope. A full disclosure of such compounds is provided in copending Application Serial No. 08/679,671, which is incorporated by reference herein in its entirety.

When the liquid to be microtagged is a petroleum or hydrocarbon, the chemical compound may be a phase transfer catalyst. Phase transfer catalysts such as crown ethers, cryptands, tetraalkylammonium salts, tetraalkylphosphonium salts and similar compounds may be used to

- 5 - dissolve radioactive iodide or other salts containing radio-elements compatible with multiphoton detection and thus label hydrocarbon mixtures such as crude oil, gasoline and other petrochemical products for later identification.

A radiolabeled microtag according to the present invention may be detected directly by multiphoton detection. Where the microtag is a chemical compound derivatized to a radiolabel, the microtag may also be detected and concentrated using multiphoton-enhanced chromatography, also described in copending Application Serial No. 08/679,671.

Chromatography enhanced by multiphoton detection according to the invention is up to four orders of magnitude more sensitive than previous techniques using radioisotopes for quantitation of chromatography outputs. Chromatographic methods and reagents according to the invention are suitable for the detection and quantification of a chemical compound or analyte of interest present in a liquid at ultralow concentrations, such as one attomole per ml (one femtomolar, or 10^"15 M), or less than one part per trillion, and in some cases below one part per quadrillion. Chromatography according to the invention comprises any technique for separating components of a mixture involving differential migration of the components through a medium, based on the physical-chemical characteristics of the components. Thus, chromatography includes liquid chromatography (LC), thin layer chromatography (TLC), gas chromatography (GC), ion exchange chromatography (IEC), capillary electrophoresis (CE), high pressure liquid chromatography (HPLC), gel electrophoresis, affinity chromatography, and other methods known in the art.

When radiolabeled chemical compounds or analytes are used as microtags, the method of the present invention comprises providing a radiophore, meaning a radioactive derivatizing agent comprising a multiphoton-emitting radioisotope moiety and a moiety reactive with the analyte of interest, the radioisotope moiety being bound to the derivatizing agent by a bond that is stable under the conditions employed in the other steps of the method; derivatizing the analyte of interest with the derivatizing agent; microtagging the liquid with the derivatized analyte; possibly and subsequently separating the analyte of interest from other components of the liquid by chromatography; and detecting the analyte of interest using multiphoton detection. Derivatization with the radiophore may be performed before the analyte is used in a method of microtagging, or after chromatographic fractionation. If derivatizing is done post-

- 6 - fractionation, it is necessary to remove excess non-reacted derivatizing agent prior to multiphoton detection, for example by means of a solid phase reagent that binds non-reacted derivatizing agent and can be filtered away from the analyte of interest.

The radioisotope is bound to the derivatizing agent to produce a radioactive derivatizing agent, preferably prior to derivatization of the analyte of interest. The radioisotope-derivatizing agent bond is stable under anticipated reaction conditions, and is preferably covalent. As should be clear to those of ordinary skill in the art, the derivatizing agent is selected depending on the class of compound to be derivatized.

The derivatizing agents may contain two or more multiphoton detection compatible radioactive atoms in one molecule, providing amplification of the signal which can be used to further increase the sensitivity of multiphoton detection enhanced chromatography.

Alternatively, multiple color analysis may be performed with radioisotopes applied via multiple derivatizing agents reactive with different functional groups of the analyte of interest.

It should be noted that a mixture of different radioisotopes used simultaneously in the same microtag may act as a signal means to indicate that the liquid has been microtagged, but may also serve as the code itself by distinguishing the liquid from other liquids which are tagged with a different combination or ratio of radioisotopes.

Where the microtag is a nucleic acid, the method of the present invention may also combine multiphoton detection of radioisotopes with PCR. When PCR analysis is performed with radiolabeled nucleotides, multiphoton detection analysis dramatically increases the sensitivity of the assay. A positive PCR reaction may be detected before saturation, i.e. less than 20 cycles of

PCR are used in the amplification process. For example, PCR may be stopped after less than ten

PCR cycles using subsequent multiphoton detection, whereas a typical PCR-based detection assay might require at least 20-30 cycles. It should be apparent from the present disclosure that the particular types of microtags described herein may be combined and used simultaneously, both as a way to create a coding means merely by the specific combination of microtags included, or to adapt the microtagging system to a particular material to be tagged.

For instance, where the microtag is enclosed in or attached to a particulate as defined above, the particulate itself may be further encapsulated in a microcontainer. Where the microcontainers are formed from thin films, encapsulating can be deliberately heterogeneous

- 7 - either in size or content so that the capsules may exhibit variable density in the liquid to be marked, or exhibit additional signal or coding means. Where the microcontainers are formed from thin films and no signal means is attached to the surface of the microcontainers, the detection or identification step may include rupture of the microcontainers by mechanical or chemical means.

The present invention also encompasses a liquid obtained by the microtagging procedure. Such a liquid may contain any combination of signal means and coding means according to the present invention, but preferably contains an additive comprising a plurality of particulates in an amount no greater than 1 part per million, the particulates comprising at least two stable signal means to aid their detection, the first signal means comprising a nucleic acid and the second signal means being other than a nucleic acid. In the preferred liquid, the nucleic acid signal means may also serve as a coding means, and said second signal is preferably a multiphoton-emitting radioisotope.

Also included in the present invention is an additive for microtagging a liquid, wherein said additive may comprise any combination of signal and/or coding means, but preferably contains a plurality of particulates, the particulates comprising at least two stable signal means to aid their detection, the first signal means comprising a nucleic acid and the second signal means being other than a nucleic acid. In the preferred additive, the nucleic acid signal means may also serve as a coding means. When said second signal is a radioisotope, it may be added to the additive just before the additive is added to the liquid to be microtagged.

Another aspect of the present invention is a microtagging kit comprising a predetermined library of additives, wherein each additive comprises at least one stable coding means that distinguishes that additive from all others in the kit, and at least one stable signal means, wherein said signal means may either be incorporated into each separate additive before distribution of the kit, or supplied separately to be added to each additive at the time of use. Again, it should be clear that the coding means may be conferred by a distinct combination of signal means. In the kit according to the present invention, the coding means may be contained within or bound to a particulate, and the particulate may be a microbead, microsphere, microcontainer, bacterium, or bacterial spore. Preferably the coding means is a nucleic acid contained within a bacterial spore. Where the coding means is a nucleic acid, a kit of the present invention may further comprise nucleic acid primers for subsequent detection of said nucleic acid using PCR. In

- 8 - addition, where the particulates used in the kit of the present invention are bacterial spores, the kit may further comprise medium for germination of the bacterium. Where the signal means is a radioisotope, a kit of the present invention may also be supplied with a portable Multiphoton detection device. The present invention also encompasses an apparatus for identifying microtagged liquids comprising a first means for detecting and identifying a signal means in a liquid, thereby indicating that the liquid has been microtagged with a coding means, a second means for identifying and characterizing the coding means, and an analyzer for comparing the coding means to a correlation list and determining the identity of the liquid. The apparatus may optionally comprise a means for assigning and correlating the coding means with the liquids to which they were added, and a display reporting the identity of the liquid. In addition, the apparatus may also comprise a means for sampling the liquid, and a means for concentrating the coding means in such a sample of the liquid.

For the case that the radioactive signatures discussed above are to be employed, it is necessary to have an apparatus which can distinguish the different radioisotopes being used; this apparatus will detect and distinguish various radioisotopes in order to determine their radioactive signatures, and perform the steps of analysis necessary to determine their proportions.

Finally, data processing can match the signature with the records on file, preferably electronically, thereby indicating which liquids have been marked with which signature. It should be noted that such steps of data analysis and processing are in addition to those commonly involved in the radiation detector itself, such as in the pulse shape analysis of multiphoton detection instruments or more conventional radiation counters.

The automated performance of the above steps will often be preferred. This will clearly be advantageous in large complex installations, allowing the instrumentation to be placed in difficult locations, and will reduce certain risks of error connected with human intervention. An apparatus for automated on-line monitoring of fluid levels or leaks in a machine or installation comprising several fluid-containing compartments should also include a means for issuing a warning when the monitoring of the signatures indicates critical levels of wear or damage, and furthermore a means for indicating which compartment is in question by identifying and correlating the signature to the particular compartment.

- 9 Further objectives and advantages will become apparent from a consideration of the description.

DETAILED DESCRIPTION OF THE INVENTION In describing preferred embodiments of the present invention illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following concepts and terms are defined below.

In the context of the present invention, the term "microtagging" means marking a material with a tag that is not visible by human sight alone, but that may be used in conjunction with other tools for the purpose of identifying the material. Accordingly, a "microtag" is the unobtrusive marker that is used to tag the material.

The term "material" may denote a solid or fluid, with the term "fluid" representing a gas or a liquid, or finely divided powdered or ganular media. The term "liquid" should be construed sufficiently broadly to encompass viscous and semisolid materials, such as tars and resins.

"Extreme dilution" indicates a dilution of at least 1 part to one million.

In the present invention, a "signal means" refers to any type of mark either disclosed in or obvious in view of the disclosure which may be added to, attached to, mixed into etc. a material and subsequently detected, thereby distinguishing the marked material from the same material which has not been marked. A "coding means" is differentiated from a signal means only in the specificity of the mark to the marked material. For example, if the population of materials to be marked and differentiated is small, a coding means may reside in the mere presence of a signal means, or in a combination or ratio of signal means. Alternatively, if the population to be marked and differentiated is large, the signal means may serve merely as a preliminary test which reveals (1) that the material is marked or contains a coding means; and (2) what type of coding means is present.

- 10 - The term "stable" when used to describe the signal or coding means indicates that the microtag remains intact and detectable during the useful life of the tagged material under the conditions of transport and use. Accordingly, for a radioisotope, "stable" means that the half-life must be suitably long depending on the time period after which the signal or code must be detected.

A microtag may be any chemical compound or combination of compounds which may be directly detected, which includes radioisotopes. Alternatively, the microtag may be housed or displayed on a particulate.

In the context of the present invention, a "particulate" is a micron or sub-micron sized particle or particulate material (as opposed to a sub-atomic particle). A particulate may be living or non-living, and may be construed as a type of vehicle or compartment that may stabilize, protect or combine the various signal means and coding means of the present invention. When the particulate is living, it is preferably a "microorganism", which means any unicellular organism or the like, including bacteria, bacterial spores, yeast, algae, viruses, etc., but it is conceivable that such a particulate could also be a cell derived from a multicellular organism.

A brief survey of types of materials suitable for providing the "forensic" information on or within microtags is useful before proceeding with the description of the invention. Tags can be usefully classified as active, excitable, passive and encoding:

(1) Active tags emit signals without any external stimulus. The simplest examples are radioactive materials whose emissions cannot be modified.

(2) Excitable tags can be externally stimulated to emit an identifying signal. "Invisible" ink is a classical use of a macroscopic excitable tag. More modern examples are fluorescing substances, which convert higher energy incident photons to characteristic emission spectra in the visible or infrared ranges. Also, some isotopes are suitable because of the feasibility of stimulating nuclear excitations or transmutations with resultant, characteristic gamma photon emissions. These isotopes include in particular the rich family of lanthanides. The stimuli for gamma emission may be other gamma rays or neutrons.

(3) Passive tags are substances which are neither emitters or excitable directly, but whose presence can be ascertained by appropriate instrumentation and/or assay systems. Magnetic labels are an obvious example. Less obviously, environmentally rare, stable isotopes can be embedded in products and later analyzed for by mass spectroscopy. Substances which have served as

- 11 - antigens by eliciting antibodies can serve as passive tags, as they can be can later be scored for by any of a variety of immunoassay procedures. A great utility is their diversity. Immunoassays can distinguish of the order of millions of antigens, thereby providing for discrimination of a huge number of forensic reporters. (4) Encoding tags refers to the use of symbol systems such as known language or cryptography codes. The symbols may be formed from active, excitable or passive materials, and also the more ephemeral electronic and electromagnetic transmissions. One obvious advantage of encoding systems for microtagging is the great amount of information which can be stored compactly wim a few distinct symbols. With respect to the size of symbols, the capacity to array single atoms into symbols and read them out has in the last few years been achieved through technologies of atomic force microscopy. However, this process is not yet practical for broad scale applications, especially if portable instrumentation is required. Furthermore, atomic force microscopy is limited to solids only, and is not very useful for encoding fuels or other liquids.

A description of the utilizations of active, excitable, passive and encoding tags supporting these needs is first disclosed, followed by disclosure of methods of fabrication and some new implementations. Each of the forensic elements described may not be necessary for each microtagging application.

With respect to active tags, an innovative use of radioisotopes is one constituent of this invention. Any addition of radioisotopes to the environment is generally considered undesirable.

However, there has recently been described a new radiation detector, the multi-photon detector (known by the trademark MPD of BioTraces, Inc.) (U. S. Patent No. 5,532,122 and U.S. Application Serial No. 08/669,970, incorporated by reference herein) which allows traces of particular isotopes to be detected at levels far below that of the environmental radiation background. This background is of the order of events per second, as reported by radiation survey meters sensitive to all types of ionizing events. In inorganic materials the background is predominantly due to radon, cosmic rays and the radioactive K⁴⁰ isotope of potassium. In organic materials it tends to be dominated by the natural abundance of C¹⁴ and K⁴⁰. The MPD multiphoton detector has the capability to discriminate against the environmental background by selectively rejecting single gamma and beta radiation background and thus increasing the sensitivity for members of the electron capture (EC) and positron-gamma emitting (PG) families

- 12 - of isotopes. These isotopes decay with emission of multiple coincident photons. The MPD multiphoton detector only reports events which have multi-photon decay signatures indistinguishable from the target EC or PG isotopes. The background registered by the portable MPD multi-photon detector is only a few counts per day, when optimized for particular EC or PG isotopes. The MPD multi-photon detector has exceptional dynamic range, at least a billion fold, allowing samples to be quantitated without dilution over the activity range of a few counts per day to 10,000 counts per second.

The use of ultralow quantities of the isotopes coupled with the capability to quantitate sub- background activities makes microtagging environmentally acceptable and economically practicable. This approach permits background rejection to 0.001 pCi (10^"14 Ci) or about 1 count per day. For singly radiolabeled molecules it permits the quantitation at the 0.1 attomole (10^"19 mole) level. In a test of microtagging according to the invention, I¹²⁵ labeled petroleum can be detected after a 1: 100,000,000,000 dilution. In this experiment, the isotope was combined with a crown ether, which was then diluted in petroleum. The resulting mixture was assayed and detected at the minute concentration resulting from the enormous dilution stated above.

Thus the EC and PG isotopes can be used as innovative tracers. The amounts necessary are far below those required for traditional tracers, permitting activities far below levels considered hazardous, either in fact or by law. The MPD multi-photon detector can also distinguish a plurality of co-resident EC and PG isotopes using the characteristic energy of their nuclear gamma emissions, which is useful for dating purposes.

Innovative, ultrahigh sensitivity isotopic dating can be implemented with EC and PG isotopes. When (at least) two radioisotopes with their different half lives are co-resident in/on a material, the ratio of their current activities suffices for calculation of their past ratios. With their relative abundances specified at the time of a microtag fabrication, and the time of subsequent addition to a carrier bulk material also known, the "age" of bulk materials in transit is simply calculated. This unfalsifiable "isotopic dating" can be compared with the claimed manufacturer's / shipper's age and quite independentiy compared with the age specified in coding constituents of a microtag. For microtagging purposes, e.g. for use as tracers and clocks, the iodine isotopes I¹²⁴, I¹²⁵ and I¹²⁶ are very simply used. They can be readily adducted to double covalent bonds of particulate components of a microtag. Other useful families of PG and EC isotopes include heavy metals and d e lanthanides.

- 13 - With respect to excitable tags according to the invention, the use of fluors in combination with PG/EC isotopes provides additional utility in microtags permitting estimation of the lifetime of die product. For example, stable, fluorescing lanthanide isotopes can serve to report the quantity of the lanthanides used in formulation of a microtag, and to serve as a normalization factor for radioactive lanthanides whose quantity decreases exponentially with time.

With respect to passive tags a plurality of innovative uses are disclosed. One is the use of immunologic techniques for microtagging purposes. We disclose herein the use of many families of antigens as microtags. More specifically, die rich family of immunoglobulins (IgA, IgG, ...) can be used as passive tags. Similarly, mere are very rich families of organic allergens for which monoclonal antibodies are available. We disclose how this large class of antigens can be used as microtags. The use of a biotin + streptavidin + I¹²⁵ reporting strategy to provide the highest sensitivity reporting is disclosed.

Existing art allows the raising of antibodies against both natural macromolecules, synthetic polymers and small organic chemicals which can be adducted to macromolecules. Microtag surfaces can have or be equipped with a variety of antigenic groups. The complementary antibodies can promote agglutination of microtags to achieve a highly specific microtag purification from complex mixtures. Antibodies can also be labeled with fluorescent adducts. They then serve to selectively display microtags among an excess of other materials by immuno- fluorescence methodologies. Such fluorescence reporting from several distinct antigens on a microtag can serve as a simple preliminary classification of the possible origins, prior to the more complex readouts requiring microtag rupture which are described below. Antigenic structures on a microtag can be considered a type of coding information, which is however much less dense than me polymer codings described below. Highly specific binding can also be accomplished with other pairs of reactants. For example, biotinyl adducts on a microtag surface would be captured by steptavidin. Chromatography columns packed with streptavidin coated beads are commonly used in molecular biology for isolation of biotinylated molecules. They could similarly serve for capture of biotinylated microtags. Desired bindings could also be implemented by linking immunoglobulins to a microtag surface, so that the complementary antigens on a solid phase could later be used to capture die microtags. The advantages of the use of antibodies versus antigens is highly application specific.

Generally, stable small antigenic epitopes are readily available and are suitable. Some of them,

- 14 - e.g. some of the pesticides, fungicides and herbicides are available which are stable even in water solutions and are not removed by aquatic microorganisms. However, there may be some level of cross-talk between different antigens in the same family. Thus, for simple, small antigens, the corresponding antibody's immunological specificity may be lower than for large molecules such as immunoglobulins. Antibodies are huge molecules, highly unstable outside die body or test tube mimics thereof. Antibodies are thus not suitable for use in general environments for microtag use and their costs would be considerably higher than for simple antigens. In some applications, however, the use of more specific coding by a plurality of antibodies may be considered. When using antigens as encoding microtags, a plurality of reporting strategies may be used. The first class of applications are those when the dilution is much less than one part per billion level. Highly sensitive immunologic assays are not necessary for such dilutions when the microtags have surfaces which are densely coated with antigens. This is not a high sensitivity immunological issue and even an agglutination tests can be used. Agglutination is d e grossest of immunologic assays but is very easy to detect optically and thus may be effective at part per million dilutions. Fluorescence reported immunoassay can be used when the number of distinguishable tags is less than four. Immunoassays have been developed witii sensitivity as high as 1 pg/ml using both EIA (enzymatic immunoassay), FIA (fluoroimmunoassay), and RIA (radioimmunoassay). Unfortunately, the prior-art instrumentation used in EIA, FIA and RIA is not portable. Also, when bacteria or their germs are used as microtags, display can be routinely done with appropriate stains using microscopes (see Fluorescent in-situ Hybridization [FISH] techniques).

The use of MPD multi-photon detectors is advantageous and permits the quantitation of competitive assays at much less tiian 10^"12 g/ml using portable devices. This translates into an ability to quantitate down to 10^"16 mole for typical antibodies/antigens. Thus, the use of a multiphoton detector for detection of antigens /antibodies in appropriate encoding microtags is disclosed. Further, signal amplification is possible by using biotin + streptavidin + metallothionine + EC/PG isotopes and is disclosed herein. In many applications, detectability by EC/PG labeling is sufficient and is the preferred implementation. Additionally, the use of multicolor capability of Multiphoton detection-assay for these families of immunological agents used as microtags is claimed. The use of said families of antigens/antibodies permits me design

- 15 - and implementation of encoding microtags. However, in the applications when dilutions are higher than one part in 10¹², me application of more complex processes which require prior concentration of microtags may be necessary. Such uses are disclosed in die present invention. At the level of relatively simple molecules, a practical coding system is available. For example, nucleic acids can serve as very densely coded forensic substances. Distinguishing elements of the natural genetic code are the adenine, thymine, cytidine, and guanine bases of deoxyribonucleic acids (DNA). They have a variety of natural and synthetic analogues which can similarly serve. These include die peptide backbone nucleic acid which are stable against degradative enzymatic attacks by natural nucleases and are more stable in duplexes for lack of a negatively charged phosphodiester backbone (Egholm, et al, Nature, Vol. 365, p. 566).

Understanding of nucleic acids is now rich enough to implement their use as a forensic code. Nucleic acid segments of any desired sequence can either be produced synthetically or as alterations to an existing DNA. With respect to coding capacity, it suffices to mention that a DNA segment of 15 subunits has about a billion different possible sequences of its four constituent subunits. This is enough to discriminate products and tiieir means of transportation important for commerce long into the future. Producing such segments with chosen flanking sequences is easily within the current arts of DNA chemistry and the constituent steps are in daily practice in biomedical and biotechnology laboratories. In one of the disclosed implementations, the DNA fragments are to be induced into micron size containers, e.g. microporous beads. These forensic DNA segments can be also established in a variety of microbes, through current techniques of genetic engineering. Therein they can be replicated with their host, protected against mutagenic damage by chromosomal repair systems and be protected against harsh treatments by the tough coat of the microbe's sporulated form. In principle any polymer comprised of a few subunits could be similarly utilized as a coding system. But synthetic polymers currentiy lack the particular advantages just mentioned and are further not suitable for forensic DNA recovery through the polymerase chain reaction (PCR) or other competitive DNA recovery schemes.

The most important feature of PCR related technologies for microtagging is that even single DNA molecules can be amplified into quantities suitable for detailed display by DNA sequencing. Moreover, selective PCR amplification of a desired DNA segment can be accomplished in an environment abundant with contaminating DNAs, so long as primer sites

- 16 - flanking the ends of die sought segment are unique among all the DNAs present. Thus PCR can be implemented to amplify and recover forensic DNAs of microtags even when there remains substantial contamination witii DNA from otiier sources.

More specifically, the use of PCR and of otiier exponential amplification schemes will be very useful in die detection of die above disclosed biological microtags. There is obviously the well known problem of saturation of exponential amplification schemes which makes quantitative PCR very difficult, especially for a large number of distinguishable DNA fragments amplified concurrently. The use of a MPD multi-photon detector with its exquisite sensitivity is highly useful because it permits quantitation of PCR products after only a few steps of amplification rather than after the 20 and more steps used in prior art. It should be pointed out that PCR does not saturate until amplification of about a few thousand is achieved, i.e. the use of less than 10 cycles of PCR amplification permits true unbiased quantitation of the DNA fragments. Thus MPD multi-photon detectors can achieve quantitative PCR of DNA-based microtags.

A variety of physical materials can serve for the particulate components of microtags, in which to house or mount the forensic information. A dense array of bubbles with sizes down to the 10 micron range and containing forensic molecules can be formed from paired tiiin films. These arrays can subsequentiy be sliced into microscopic envelopes ("confetti"). The component chemicals of sealant and films can be chosen so that they will be stable in the natural environment and the bulk materials to be microtagged, while providing for a simple rupture by a chosen chemical treatment.

Microporous beads initially developed for purposes of chromatography can house forensic materials. For uses such as microtagging drugs, microporous bead materials may often be preferable. They could be encapsulated with thin indigestible films and thus be harmlessly passed dirough the digestive tract if ingested. Using less than 50 microns diameter, non-visible microtag components is aesthetically most desirable for these applications.

The use of micron sized beads or enclosures is complicated by the presence of other particulates, especially in die case of spills in the aquatic environment. Some innovative implementations of differential filtering and enhancing microtags concentration are described below. Possibly d e simplest is the use of microbeads or microspheres, and enclosures witii significant magnetic permeability. If the liquid containing such microtags is placed in a strong magnetic field, die microtags will be attracted magnetically towards the magnet, permitting low

- 17 - cost differential filtration. There are three physical implementations of such magnetically enhanced microtags. In the first, die physical core of a microtag is a micro-crystal of magnetite or other material with large magnetic permeability. This magnetic kernel is coated witii porous encapsulating polymer as described below which houses die forensic information, as well as bringing the finished microtag to a density appropriate for suspension in bulk liquids. In die second, material from which microbeads are produced, e.g. porous plastic, is seeded randomly witii submicron size grains of material with high magnetic permeability. Finally, the nonmagnetic microbeads can be externally coated with thin layer of me magnetic material, e.g. plated witii very thin layer of metallic ferromagnet. For environmental applications microbe-based microtags would have multiple benefits.

Methods of introducing the DNA segments into microbial hosts are well established. The microbes thus engineered can be expanded into huge populations at low cost. Population densities of a billion bacteria per ml and 100 billion viruses per ml are easily attained. Many suitable microbes form extremely resilient spores which can sustain exposure to otherwise toxic chemicals, lethal temperatures or dehydration and still retain viability. To prevent spores from an undesirable early germination, microbes witii a nutritional deficiency can be used, so that germination can be indefinitely postponed until these spore microtags are recovered and placed in me proper nutrient environment. In die course of germination, any damage to single strands of duplex DNA is typically repaired. Current skills of genetic engineering easily permit alteration of surface antigenic features of microbes, to allow their discrimination from more abundant relatives in the natural environment. Lastly, many microbes are motile and will preferentially swim into beneficial environments and away from noxious environments. Such natural or genetically engineered trophisms can serve if necessary for purifying and concentrating microbes. In particular motile bacteria developed from spore components of microtags can thus be gathered. Microbes are pervasive and expected occupants of almost all environments. Hence those serving as microtags will not be simply recognizable among much more abundant environmental microbes. Their presence will not be a hindrance to most intended uses of bulk goods. Except for drug and some food products, in most manufacturing there is no dedicated effort to bar trace microbes. Thus the use of microbes as microtags hides their purpose and poses a severe hindrance to attempts to confuse die origins of die bulk materials seeded.

18 - For any particular core of a microtag, there can be advantages of adding additional layers. For example, microbial spores would by memselves tend to sediment through petroleum, because they are denser and have hydrophilic surfaces. Micro-encapsulation in a less dense, crosslinked polymer can beneficially modify these characteristics. The encapsulating can be deliberately heterogeneous so that some of the capsules will: float on the oil surface; be wetted and ti ereby mix well with petroleum; sink through oil but float on water; or sediment through water, to be retained in the tanker's bilge or on the sea bottom in the case of spill. Thus a capsular heterogeneity provides optimized opportunities for marking the original spill site, deducing the subsequent drift caused by tides or river flow, as well as the multiple microtagging of the carrier vessel(s) itself. The same benefits of encapsulation are applicable to micro-envelopes or porous beads.

Encapsulating layers have several other utilities. They can house additional forensic components. Concentration of dispersed microtags will sometimes be desirable to facilitate readout of forensic information. Differential sedimentation and/or differential filtration are two suitable concentration processes. In the case of microtags of microbial size, an overwhelming abundance of otiier microbes would be concomitantly gathered. By increasing the original microbe size a few fold, accumulation of most of this unwanted community can be substantially minimized. The encapsulating can serve as a source of antigens not commonly found in the environment. These antigens will support microtag purifications through aforementioned immunological techniques.

With respect to chemical properties the capsular material can be chosen so that it can be dissolved by a chosen solvent which is neither present in d e tagged bulk material or in potential spill environments. Such choices are similarly useful for microtags fabricated from thin films. They will allow the simple exposure of the forensic molecules (antigens, DNA, PNA, etc.) for immunoassays or sequence analysis. For example, there are polymers which are neither soluble in petroleum or aqueous environments which however dissolve in alcohol.

For non-microbial microtags, the structural materials can be chosen so that tiiey will dissolve or rupture when treated witii solvents. The structure solvent combinations are chosen so that the solvents will not be encountered in me environments of microtag usage. For any type of microtag there are several methodologies of mechanical rupture which can serve to release forensic molecules. These include freeze fracture metiiods, the French Pressure Cell designed

- 19 - for breakage of microbes as small as bacteria and simple grinding witii abrasive such as alumna. The forensic molecules, e.g. DNA segments are so small that their population will not be significantly damaged. After simple centrifugal sedimentation of particulate debris, DNAs can be prepared for analysis of their forensic components by established methodologies. Conventional techniques are applicable to detection of tags whose concentration is fixed during die manufacturing process, providing for readout of the "forensic" information with parts per million sensitivity. They are not sensitive enough to detect less than parts per billion microtags dilutions or femtomoles of microtag per liter. They are thus not applicable to the more stringent applications targeted by this invention, in which femtomole/ml tag concentrations are desirable. A further technical limitation is commonly a lack of significant dynamical range.

Typically, two different forensic materials read out by common instrumentation are distinguishable reliably only if their concentrations are within one to two orders of magnitude of one another. Some forensic components of the microtags and concomitant reporting system described herein have proven discrimination capabilities over 100,000 fold concentration ranges. For the purpose of tagging bulk materials with minute quantities of coding tags at extreme dilutions, the use of EC/PG emitters and a family of reporting/forensic materials has been designed which can be housed in a micro-particulate structure, the microtag. The reporting materials elements have synergistic roles in the overall microtag function.

Active microtags using EC/PG emitters have the highest sensitivity (down to 10^"20 mole) and very high dynamic range (over nine decades from 10^"20 to 10^"11 mole) but may be difficult to implement when a very large number of different components or products are to be tagged.

Passive and encoding microtag components are useful for discrimination and isolation of microtags from complex mixtures, through their chosen properties in binding systems. They can serve in antigen-antibody reactions. Biotinylated surface adducts will be gathered/captured by avidin or streptavidin proteins anchored to solid surfaces. Fluorogenic labels specific for different surface features can thus be applied to die microtags to support their discrimination within complex mixtures or other particulates, potentially including microtags previously contaminating an environment. Actually, in many applications the encoding with antigens or DNA/PNA combined witii reporting via multi-photon detector technique is optimal. Appropriate fluorogenic labels according to d e present invention include allophycocyanine, phycocyanine, phycoerythrine, rhodamine, oxazine, coumarin, fluorescein

- 20 - derivatives, Texas red, acridine yellow/orange, etiiidium bromide, propidium iodide, bis- benzamide, or any otiier suitable fluorescent dye that can be conjugated to die microtags or the detection tools (i.e. antibodies) of the present invention.

The nucleic acid microtags of the present invention may be DNA, PNA or RNA. A recent discussion of die characteristics and synthesis of PNA, or peptide nucleic acid, and the use of

PNA in PCR reactions may be found in U.S. Patent No. 5,656,461, Demers et al. However, the preferred nucleic acid microtag consists of DNA. To achieve the highest coding density, DNAs with a four letter code are used. In die DNA-based implementation, the coding segment is flanked by two sequence regions suitable as primer sites for the PCR reaction. The DNA microtags should be of sufficient length such that all materials of a particular class or group to be microtagged may be marked witii a different sequence. In addition, the nucleic acid microtags should be of an appropriate length to allow detection and analysis using PCR, hybridization or sequencing. Preferably, the nucleic acid is between 20 and 1000 nucleotides long depending on the type and quantity of materials to be microtagged. The DNAs can eitiier be housed in minute envelopes, microporous beads or microbes.

Microbeads/spheres are commercially available from Dynal (U.K.) Ltd. of Wirral, Merseyside, U.K. under the trade names DYNABEADS and DYNASPHERES. The microbial host system provides for economical replication of the coding DNA, and provides a source of antigenic reporting elements as well. A preferred microbial host is any species of the genus Bacillus, i.e. B. subtilis. Methods for growing B. subtilis, and appropriate cloning vectors for introducing the nucleic acid tags of the present invention are well-known in the art; see for example Youngman et al. , "Methods for Genetic Manipulation, Cloning, and Functional Analysis of Sporulation Genes in Bacillus subtilis 7 in Regulation of Procaryotic Development, Smith ed. ASM Washington, 1989, pp. 65-87 ; and Youngman, "Plasmid vectors for recovering and exploiting Tn927 transpositions in Bacillus and other Gram-positive bacteria" in Plasmid - A Practical

Approach, Hardy ed. IRL Press, Oxford, England (1987) pp. 79-203. Suitable linkers and adaptors for cloning nucleic acid microtags into a replicatable plasmid vehicle appropriate for Bacillus are available from a variety of vendors (e.g. New England Biolabs, Beverley, Massachusetts), and mediods for cloning DNA into appropriate plasmids using such linkers are well known in the art (Sambrook et al., 1989). Microbes may be equipped with genetically

- 21 selective features such as drug resistance to support their selective growth and purification away from other microbes.

The particulates of the present invention may be further encapsulated into microscopic envelopes. Methods of making such envelopes are known in the art. For example, the signals can be laminated in sheets and cut into "confetti." Encapsulation of the particulate components or bacterial spores adds functionality by:

• enclosing and retaining the reporting elements;

• conferring a range of densities, thereby providing for a multiphasic distribution of microtags in the case of spillage of the bulk material; • allowing designed hydrophilic or hydrophobic interactions between die microtag and its original and potential spill environments;

• increasing the size of the finished microtag so that they will be in a size range larger tiian most naturally occurring particulates, e.g. bacteria, thereby providing for an improved fractionation away from naturally occurring particulates by either differential filtration or differential sedimentation processes;

• providing additional surface structures to support purification by selective bindings, including antigenic sites that can serve in highly selective immunological purifications of microtags or their specific visualization within complex mixtures by immuno fluorescence microscopy; • providing sites for attachment and or embedding of otiier reporting and forensic elements. Additional reporting elements include isotopes in die EC and PG families, which can be selectively detected far below the environmental background radiation. Thus they serve as non- hazardous tracers. Particular combinations of radioisotopes fulfill date specification roles, e.g. permit implementation of isotopes as encoding components.

Although d e isotopes of the present invention are preferably detected using multiphoton detection (U.S. Patent 5,532,122), depending on d e ddution of the microtag and die strength of the isotopic signal, labels may also be detected using a Geiger-Mϋller tube, scintillation counter, or photographic film. Radiolabels may be added directly to the liquid to be microtagged, or may first be attached to particulates. The attachment of radiolabels to particulates is well known

22 After serving for the initial detection of dispersed microtags, the radiations from EC or

PG isotopes can serve to assay the progress of microtag concentration. These methodologies can include differential sedimentation or centrifugation; differential filtration and highly selective immunological purifications. Once concentrated and partially purified, die low density information of the microtag can be read out by immuno-fluorescence microscopy.

To access the information contained in densely coded polymers such as DNA, microtags are physically ruptured and die DNA component purified. Mediods for purifying DNA away from other cellular components are well known in the art (Sambrook et al.). The forensic DNA segment(s) may be selectively amplified through PCR or detected using hybridization, and further distinguished by sequencing. Standard methods for hybridization and sequencing are well-known in the art, and may be found in any molecular biology laboratory manual (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). PCR reactions may be performed using Taq polymerase (Promega, Madison, Wisconsin) according to manufacturer's instructions. The total coding information thus obtained serves in identifying the origins of the bulk material in which the microtags were originally dispersed.

These implementations of encoding microtags are an innovative combination of state of the art techniques from very different fields of cryptography, microfabrication, microbiology, molecular biology and nuclear instrumentation. This combination of diverse techniques is highly non-obvious and has been combined to provide increased sensitivity and specificity which cannot be achieved with any subset of these techniques. To further elucidate the application of the subject invention, it is useful to describe in more detail the application of the encoded biological microtag concept for the case of microtagging petroleum and subsequent detection of the source of maritime petroleum spdls. This application is characterized by the need to detect extremely low amounts of identifying microtag. Because of the large volume of the transported material it is not economically justifiable to use microtags which have a volume fraction much larger than about a part per billion. Furthermore, typically the leak or spill of this material in a maritime environment leads to a further part dilution which can be as high as an additional part per million.

When forensic information is encoded in large biological and syntiietic macromolecules, e.g. DNA, PNA and antigens tiiat are unstable in an environment of liquids such as water or petroleum, they are preferably dispersed in appropriate micro-containers. To be economical, only very low amounts can be used. For example, one may apply about 100 to 1000

- 23 - microcontainers/ml, while the natural particulates, e.g. dust, is present in about 100 million/ml quantities. Thus, the challenge is selection of the information carrier (artificial microcontainers) from naturally occurring background (dust, tar balls, bacteria/spores). This requires a two stage strategy of information noise rejection: (1) selecting the micro-containers from naturally occurring particulates; and (2) analyzing forensic information contained in the microcontainers. The first step permits about thousand-fold enrichment/purification. In the second step, the information encoded in macromolecules contained in d e microcontainers is read-out and analyzed.

To reject information noise due to die presence of a natural abundance of such molecules, a multiple vector, highly redundant system is preferred. Thus, the same information is encoded eidier as a plurality of separated information rich regions of the molecules or by using a plurality of molecules with the same information rich regions. Preferably, at least ten different methods of encryption may be used for the same information in order to suppress information noise due to naturally occurring macromolecules.

The same information can be encoded in a plurality of DNA or PNA molecules, each of diverse lengtii. Stringent selection rules can be used, for example each DNA fragment is designed to contain only an even number of the regions. Thus, redundancy techniques simtiar to "parity checking" but with a totally different physical realization may be used to achieve an increased signal/noise ratio. The same information is carried and/or encoded in different microcontainers or molecules. Only the concurrence of these messages establishes the validity of the forensic information.

Redundant information retrieval may increase the cost and diminish the throughput. Thus, before a potentially costly decoding protocol is initiated, it is important to make sure that die given fluid is microtagged. Thus, the full implementation of a mictrotagging system advantageously consists of at least two parts, a "signal" and "code itself". Thus the presence of a given EC/PG isotope may be a "signal" providing die information in which material is implemented "code itself. For example, presence of I¹²⁵ may signal that the code is implemented using DNA, while the presence of I¹²⁶ signals that the code uses PNA. With the availability of about 30 EC isotopes, there is a large versatility in such a two independent encoding systems scheme. The information system of the invention is quite different tiian the typical information theory situation. There, a large number of messages are sent by a single source or a small

- 24 - number of sources. The noise can be described as a single source, e.g. white noise, flitter noise, 1/f noise. Even in the most unfavorable cases there are no more than three or four noise sources which can be well described statistically.

In the case of microtagging, there is only one, relatively low information content code message (and me signal). In contrast, the sources of noise are both extremely variable and much more frequent than signal carrier. For example, if information is encoded in a few EC/PG radioisotopes, the natural radioactive background can be a thousand times higher and may consist of hundreds of sources, each source being a distinguishable gamma or beta emitter. In the case of using forensic information encoded in DNA, there are billions of different DNA background sources due to naturally occurring organisms. In die case of encoding information inside of the bacteria spores, there are hundreds of thousands of types of bacteria which may be present in the water or petroleum.

The following is an implementation of microtags applied for the audit trail of hydrocarbons and their chemical derivatives, including spill/leaks of petroleum or petroleum derivatives from transportation vessels, e.g. ships or barges, uses forensic DNA enclosed in spores of bacteria.

In all stages of preparation up to seeding die bulk cargo, samples are curated for purposes of later comparison with me seeded microtags. Samples of the crude petroleum and its container are tested for die presence of any other tagging materials which could be confused witii the reporting elements of the microtag. To achieve dispersion in petroleum, the microtags are first thoroughly mixed into a small volume to achieve wetting of their surfaces. This small volume is tiien added to me shipping vessel, preferable before or as the vessel is filled to further mixing and dispersion. The motions any transport vessel confers to its liquids will further help keep microtags dispersed. A sporulating and DNA transformable strain of the non-pati ogenic bacteria, Bacillus subtilus is used as a the microbial host for insertion of DNA coding information. This information will encode the identity and route of the transporting vessel for petroleum. The microbial population is expanded tiirough normal growtii. The culture proceeds into sporulation and die microbial population is harvested, rinsed and lightly dried. Iodination with tracer quantities of radioisotopes I¹²⁴ and I¹²⁵ is performed shortly before the planned dispersal of the

- 25 microtags. Half of the population is not iodinated to minimize damage to spore and forensic DNAs by decaying isotope. The two portions are mixed prior to encapsulation.

Encapsulation begins by suspending die spores in a hydrophobic polymerizing latex, for which antibody has already been prepared. A portion is not coated, to facilitate spore sinking in the vessel or spill sites. The mixture is forced through pores in die 1-10 micron size range, with formation of a range of capsule droplet sizes and accompanying densities range. The droplets fall upon stirred water where tiiey discretely complete their hardening. A non-encapsulated portion is added to die main stock. These spores will preferentially sink in the carrier vessel or at spill sites. At any spill site, harvesting and purification procedures for the microtags are then implemented. The oil film is skimmed up and sediment samples also taken. Differential sedimentation, filtration and immunological coagulations are used to concentrate die microtags, with monitoring through gamma emissions reported by a MPD multi-photon detector.

All subsequent analyses are performed witii potentially matching curated microtag samples and recovered microtag samples in parallel. Comparisons of antigenic, forensic DNA and isotopic dating information serve to establish the identity or differences of a curated and microtagged population. Mechanical shredding releases spores from their soft latex capsules. Thereafter techniques of microbial processing and PCR are implemented to display die forensic information in the DNA. The disclosed methodology implements a series of steps through which microtags are first detected by their EC or PG labels and then progressively concentrated and purified. They are removed from the bulk or the oil and water dilutant with progress reported at first only by their EC and PG isotopes. In almost all cases this step will concentrate the mixture by a few orders of magnitude and will result in a sample in which the petroleum is at least a few percent of the sample volume. The same procedure will also nonspecifically concentrate any particulate matter or debris which floats on the water. This may include natural microplankton and microorganisms living in the water. Thus, the next two proposed steps are filtration of particulates by size and immunofiltration, e.g. immunoagglutination based on specific antigenic properties of the encapsulating material. These steps will only marginally, by a factor of a few, increase the concentration of the microtags in the sample. However, they specifically remove, by a few orders of magnitude, the

- 26 - particulates and organisms which are different than the microtags. The combination of these two steps is used to increase by a few orders of magnitude the microtag concentration relative to botii dillutant and nonspecific particulate background.

The capsular materials are ruptured by light grinding with an abrasive. Addition of a supportive growth medium stimulates bacterial germination. The presence of an antibiotic to which it is resistant will inhibit other microbes. After the population is thus expanded, it can be processed by standard procedures for analyses of DNA. A low speed cenfrifugation pellets large debris. A higher speed centrifugation harvests the bacteria. They are then treated witii lysozyme to rupture their own polysaccharide coats. Their DNAs are thus exposed for PCR reactions which will make the forensic segments available for DNA sequencing.

Further steps involve the preparation of the mixture for the biochemical detection of the microtags. First, by use of appropriate organic solvents, petroleum itself is washed out. Preference is given to solvents which both dissolve petroleum, especially the high viscosity bitumite components, and at die same time leads to de-encapsulation of the microtags. For example, alcohol can be used to dissolve the adhesive part of the encapsulating material. Thus, the biological material, e.g. antibody/antigen, DNA fragments or bacteria are released.

Shortly after, in a time much shorter than the denaturation time of the biological substance in alcohol, a large amount of clean water with appropriate pH is added. PCR or other amplification techniques are then implemented, followed by detection using a multi-photon detector.

In the case where die encoding tags are microbial spores into which forensic DNAs have been incorporated, two further steps may permit even higher detection specificity and sensitivity. After the de-encapsulation and hydration steps, a short incubation at the appropriate temperature leads to spore revival. Afterwards, a further selection of the tagging microorganism from particulates and dissolved organic material naturally present in seawater is accomplished using tropisms characteristic for the given class of bacteria. The revived bacteria will literally swim toward the tropism source. For example, one can use a particular class of bacteria which incorporate in their cells a submicron magnet and are very efficiently attracted towards sources of magnetic fields. Obviously, such tropisms selects intact bacteria from organic detritus and a given class of bacteria from all other viable microorganisms present in the water. Additional selectivity can be achieved if die bacteria which are concentrated and harvested in die preceding

- 27 - steps are cultured. With culturing, the exponential amplification of a given class of bacteria can be readily achieved whereas die majority of the other bacteria will not be amplified. All of the above steps permit increased selectivity and thus increase the signal to background ratio, wherein the background is any organic material naturally present in the aquatic environment. After these selective concentration steps, PCR amplification and multi-photon detection are used to report die presence of microtags.

Once fluids are tagged according to the invention, they may also be used in connection with solids. For example, a microtagged "invisible" liquid ink may be applied to paper or otiier solid products, dried, and used later to identify or authenticate the paper or solid good. All publications and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The embodiments illustrated and discussed in this specification are intended only to teach those skilled in die art die best way known to the inventors to make and use the invention. Notiiing in this specification should be considered as limiting the scope of the present invention.

Modifications and variations of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by tiiose skilled in the art in light of the above teachings. It is therefore to be understood that, witiiin the scope of the claims and their equivalents, the invention may be practiced otiierwise than as specifically described.

28

Claims

WHAT IS CLAIMED IS:

1) A method of microtagging a fluid, comprising: microtagging the fluid by adding to the fluid an additive that is stable in the fluid and comprises at least one signal means which may be detected after extreme dilution, and a coding means to aid in identification of the fluid, wherein said coding means is contained witiiin or displayed on die surface of a microorganism or is encapsulated in a microcontainer; obtaining a sample of the fluid containing said additive; detecting the presence in the fluid sample of said at least one signal means, thereby detecting that the fluid was microtagged; if a signal means is present, decoding said coding means; and correlating the coding means with the identity of die fluid previously tagged, tiiereby identifying me fluid sample.

2) The method of claim 1 , in which said fluid is a liquid.

3) The method of claim 2, wherein said at least one signal means is also contained within or displayed on die surface of a microorganism or is encapsulated in a microcontainer.

4) The method of claim 1, further comprising logging the signal means, coding means and the identity of the corresponding microtagged liquid on a correlation list at the time the fluid is microtagged.

5) The method of claim 1 , wherein said microorganism is a bacterium or a bacterial spore.

6) The method of claim 5, wherein said bacterium or spore is Bacillus subtilis.

7) The method of claim 5, wherein said coding means is a nucleic acid or a polypeptide.

29 8) The method of claim 7, wherein said microtagging step involves transforming said bacteria with said nucleic acid, or with a nucleic acid encoding said polypeptide, growing said bacteria untd sporulation occurs, and mixing said spores witii the liquid to be microtagged; and said detection step involves recovery of said spores, exposing said spores to an appropriate medium such that germination is induced, growing the germinated bacteria such that said coding means is amplified, and decoding said coding means.

9) The method of claim 8 wherein said coding means is a nucleic acid, and said detection step comprises PCR, sequencing or hybridization, or any combination thereof.

10) The method of claim 9 wherein said detection step comprises PCR, said PCR is stopped before saturation, and PCR products are analyzed using multi-photon detection.

11) The method of claim 8 wherein said coding means is a polypeptide, and said detection step comprises an enzymatic reaction or an immunoassay.

12) The method of claim 2 wherein said signal means is a radiolabel.

13) The method of claim 12 wherein said radiolabel is selected from the group consisting of EC and PG radioisotopes, and said radiolabel is detected using multi-photon detection.

14) The metiiod of claim 13 wherein said radiolabel is an isotope of iodine selected from the group consisting of I¹²³, I¹²⁴, I¹²⁵, and I¹²⁶.

15) The method of claim 1, wherein said fluid is a hydrocarbon, and wherein said signal means is a phase transfer catalyst.

16) The method of claim 15, wherein said phase transfer catalyst is derivatized with a radiophore before being added to said liquid, and detected witiiout concentration using multiphoton detection.

30 17) The method of claim 15, wherein said detection step comprises concentration and identification of said phase transfer catalyst using chromatography.

18) The method of claim 1, wherein said microorganism is further encapsulated in an outer microcontainer.

19) The method of claim 18, wherein said further encapsulation step produces a heterogenous population of microtags having different densities.

20) The method of claim 18, wherein said outer microcontainer comprises additional signal means.

21) The method of claim 18, wherein said detecting or identifying step comprises rupture of said outer microcontainer by mechanical or chemical means.

22) A microtagged fluid containing a stable additive comprising at least one signal means which may be detected after extreme dUution, and a coding means to aid in identification of the fluid, wherein said coding means is contained within or displayed on die surface of a microorganism or is encapsulated in a microcontainer.

23) The fluid of claim 22 which is a hydrocarbon, a chemical derivative of a hydrocarbon, a fuel, a perfume, or a drug or pharmaceutical.

24) The fluid of claim 22, wherein said coding means is a nucleic acid, or a polypeptide.

25) The fluid of claim 22, wherein die signal means is selected from the group consisting of a radiolabel, a fluorescent label, a chemical compound, and a first molecule having a high specificity for a second molecule.

26) The fluid of claim 24, wherein the nucleic acid is also a coding means that specifically identifies the fluid.

31 27) An additive for microtagging a fluid, said additive comprising at least one signal means which may be detected after extreme dUution, and a coding means to aid in identification of the fluid, wherein said coding means is contained witiiin or displayed on die surface of a microorganism or is encapsulated in a microcontainer.

28) The additive of claim 27, wherein said microorganisms are further enclosed in microcontainers of varying density.

29) The additive of claim 27, wherein said microorganisms are further enclosed in microcontainers comprising at least one additional signal means.

30) A microtagging kit comprising a predetermined library of additives, wherein each additive comprises at least one signal means which may be detected after extreme dUution, and a coding means to aid in identification of die fluid, wherein said coding means is contained within or displayed on die surface of a microorganism or is encapsulated in a microcontainer, and at least one nucleic acid coding means that distinguishes tiiat additive from all others in die kit, wherein said signal means may either be incorporated into each separate additive before distribution of the kit, or supplied separately to be combined witii each additive at die time of use.

31) The kit of claim 30, wherein said microorganisms are further encapsulated in microcontainers.

32) The kit of claim 31, wherein said microcontainers contain the signal means.

33) The kit of claim 30, wherein said signal means is a radioisotope, a chemical compound, a chemical compound derivatized to a radiophore, a fluorescent molecule, or a first molecule having binding affinity to a second molecule.

34) The kit of claim 30 wherein the coding is a nucleic acid or a polypeptide.

32 - 35) The kit of claim 30, wherein said microorganism is a bacterial spore, and said kit further comprises medium for germination and growth of the bacterium.

36) The kit of claim 30, wherein said signal means is a chemical compound, and said kit further comprises a derivatizing agent for labeling said chemical compound with a radiophore.

37) The kit of claim 36, wherein said chemical compound is a phase transfer catalyst.

38) The kit of claim 30, wherein said signal means is a radioisotope and said kit further comprises a portable multi-photon detector.

39) A method of preparing an additive for microtagging a fluid, comprising selecting a signal means, a coding means, and a particulate, and encapsulating within or binding to the particulate at least one of said signal means and coding means.

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