Title of Invention	"DEVICE FOR RAPID DETECTION AND IDENTIFICATION OF SINGLE MICROOGANISMS WITHOUT PRELIMINARY GROWTH"
Abstract	This invention describes a device consisting of a micro channel plate, filter, and porous holder for filter, which is substituted by a pure agar block during method performance, and supportive structural elements. The device is intended for rapid detection and/or identification of microorganisms, Microorganisms are trapped by filtration in long (diameter/length-1/10-1/1000), cylindrical, parallel, micro channels that are open from both sides and attached to a filter from one side. A micro channel plate houses a multiplicity of micro channels (possible diameter of each channel=l-30 µm length 100-1000urn and number channels per centimeter =100,000-1,000,000). The micro channel plate with cells trapped on the surface of the filter is attached to an agar block impregnated by artificial substrate(s) so that the molecules of the artificial substrates will fill all micro channels. Trapped cells produce colored or fluorescent molecules from artificial substrates. These molecules are collected in the very small volume of a micro channel. The extremely small volume of a micro channel (1/25 millionth part of milliliter) allows it to collect a detectable concentration of color or fluorescent substances in a very short time (several minutes). Even one cell from a filtrated sample can be detected by the enzyme-artificial substrate method and/or identified by enzyme immunoassay.

Title of Invention

"DEVICE FOR RAPID DETECTION AND IDENTIFICATION OF SINGLE MICROOGANISMS WITHOUT PRELIMINARY GROWTH"

Abstract

This invention describes a device consisting of a micro channel plate, filter, and porous holder for filter, which is substituted by a pure agar block during method performance, and supportive structural elements. The device is intended for rapid detection and/or identification of microorganisms, Microorganisms are trapped by filtration in long (diameter/length-1/10-1/1000), cylindrical, parallel, micro channels that are open from both sides and attached to a filter from one side. A micro channel plate houses a multiplicity of micro channels (possible diameter of each channel=l-30 µm length 100-1000urn and number channels per centimeter =100,000-1,000,000). The micro channel plate with cells trapped on the surface of the filter is attached to an agar block impregnated by artificial substrate(s) so that the molecules of the artificial substrates will fill all micro channels. Trapped cells produce colored or fluorescent molecules from artificial substrates. These molecules are collected in the very small volume of a micro channel. The extremely small volume of a micro channel (1/25 millionth part of milliliter) allows it to collect a detectable concentration of color or fluorescent substances in a very short time (several minutes). Even one cell from a filtrated sample can be detected by the enzyme-artificial substrate method and/or identified by enzyme immunoassay.

Full Text	DEVICE FOR RAPID DETECTION AND IDENTIFICATION OF SINGLE MICROORGANISMS WITHOUT PRELIMINARY GROWTH BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] Modern microbiological diagnostics and analysis serves for detection, enumeration, and identification of microorganisms present in different samples. In the areas of medical diagnostics arid veterinary medicine, these organisms are pathogenic or dangerous microbes in human or animal blood, internal organs, skin, tissues, respiratory organs, and so on. In the area of industrial microbiology, microbes commonly pollute technological processes, materials, equipment, and finished products. In environmental analysis, there is often microbial contamination of water, indoor and outdoor air, and various surfaces. In epidemiology and biodefense - highly contagious pathogenic microorganisms from human body or environment. [0002] Time, quality, arid sensitivity of microbiological analyses arc crucial for two reasons. First, tens of thousands of domestic microbiological laboratories spend several billions of dollars annually for products and processes quality control and prevention of contamination and spoilage in industry. These laboratories also spend money to provide diagnostic tests for humans, animals, plants, food, personal care products, soil, and environment. A quick, reliable, streamlined diagnostic test can save companies millions of dollars in the long run. Second, several thousands of people die even in such highly developed countries as the United States because of a delay in medical treatment caused by the length of time for diagnostics. Decreasing the time it takes to run these microbiological diagnostics tests will result in an increase of analysis reliability and sensitivity. Ultimately, this can save thousands of human lives worldwide. [0003] The present invention relates to detection and identification of microorganisms without preliminary growth. In particular, the present invention relates to detection and identification of microorganisms without preliminary growth. 2. Description of the Related Art [0004] Modern methods of detection, enumeration, and identification of microorganisms can be divided into two main parts 1) the methods and devices that need preliminary growth (enriching) to create a detectable amount of cells; 2) methods that don't need preliminary growth because they are capable of analyzing as little as a single cell. [0005] The first group includes growth in solid or liquid regular or selective nutrient medias. It also includes several immunological methods. Examples are latex and hemagglutination, antibodies on magnetic particles, enzyme immunoassays like ELISA and Western Blot, and "dipstick" methods. The first group also includes chromatography of fatty acids, infrared Raman and FTIR spectroscopy, mass-spectrometry, and ATP-, bio-, and chemiluminescence. These methods require hundreds to millions of pure cells for detection of a certain microorganism, and, therefore, long (many hours or days) preliminary growth. [0006] The second group of methods and devices does not need preliminary growth because they are capable of detecting and/or identifying even a single cell. These methods and devices consist of a group of nucleic acid methods like PCR and its various modifications, Epi-fluorescent methods (fluorogenic substrate methods, immunofluorescence), and a group of Flow Cytometry methods. [0007] In addition to other drawbacks, methods of detection and/or identification of cells without preliminary growth usually need very expensive and sophisticated equipment and the work of high-level professionals. For example, devices used for PCR include the expensive thennocycler and sophisticated fluorometer. PCR is also used only for identification purposes. Enumeration of initial contamination is not reliable with PCR usage. PCR is very sensitive to organisms which can contaminate the test itself. [0008] Epi-fluorescence usually needs only a fluorescent microscope to detect a single cell marked by a fluorescent dye or fluorescent antibody (Ab+fluorochrom). However, the amount of fluorescent substance present is restricted by volume of the cell body or cell surface. The small size of the object (single microorganism) makes the detection of one cell very difficult, especially with large background fluorescence usually present in a majority of the samples. Substances that flow out of a cell or enzyme immunoassay of a single cell are impossible with Epi- fluorescent methods because these indicator substances disperse in the surrounding space immediately but are not concentrated in a small volume like proposed by the current invention. [0009] Flow Cytometry is based on a very complicated opto-electronics system. A flow cytometer consists of a sophisticated optical block, a block of electronics, a complicated hydrodynamic system, and a high-speed computer. Prices for different types of flow cytometers range from $50,000 to $140,000. Flow cytometers can analyze one single cell during its flow through channels with diameters of 10 microns each. This size of the channel is so narrow that it needs 17 hours to pass 100 ml of liquid through it, even if speed of flow is 20 meters per second. Therefore, flow cytometers are currently used in hematology because of large (5-6 million/ml) and more or less stable concentrations of blood cells. Also, flow cytometers are very effective as sorters of cell mixtures in cytology. Usage of flow cytometry in microbiology is not easy. In microbiology these instruments are not often used because microorganisms can create clusters with other particles and can be confused with natural particles or dead cells. If the concentration of cells in a sample is very small, the time of analysis goes up dramatically. Therefore, preliminary concentration or even enriching is needed for Flow Cytometry microbiological applications. Microbes are also much more diverse by size and shape than blood cells and, thus, mistakes occur very often. [0010] It is known, and currently used in practice, that dividing a sample into small volumes helps to detect cell concentration faster. This effect depends on reaching a detectable concentration in a small volume faster than in a large volume. U.S. Pat. No. 5,716,798 describes the method for rapid detection of microorganisms in a container divided on a plurality of discrete zones, each of which can be separately monitored for microbial presence by reaching detectable cell concentrations after preliminary growth in some zones. This method gives time-saving of 10% to 40% in comparison with other methods. U.S. Pat. No. 5,770,440 is based on the same effect. The present invention differs from these patents because of the analysis of a single cell. No time-consuming preliminary growth or nutrient media are necessaiy. [0011] U.S. Pat. No. 4,959,301 is based on dividing a sample with viable biological entities into micro-droplets and detecting entities by growth or by biochemical reactions of a single entity within a droplet. This method can indicate a single cell in less than 30 minutes in some variants. Nevertheless, it is technologically complicated. Micro-droplets are produced with different volumes and require statistical analysis for calculating results. This method could be reproduced only in a laboratory by highly professional personnel with use of sophisticated and expensive equipment. [0012] The proposed device has significant advantages in comparison with known methods: [0013] It is capable of detecting and/or identifying by colored or fluorescent enzyme or enzyme-immunoassay as little as one single cell trapped in a micro-channel (one cell in one micro-channel corresponds to a concentration of 25 million cells per ml). Thus, no preliminary growth is needed, and a detectable concentration is reached in several minutes. [0014] The price of the device and analysis is ten times less than Flow Cytometry or PCR (only a regular fluorescent or light microscope is needed for this diagnostic device). Also, the amount of reagent needed is substantially less than with the use of regular 96-well plates. As a result, the analysis is simple and cost- effective. [0015] The device is simple to use and involves performing only regular filtration with just a few manipulations. Even non-specialists can easily adopt this device and procedure that is very important for their broad use. [0016] Many different methods can be applied with the proposed device: detection of live cells by fluorogenic or chromogenic substrate(s), differentiation by special enzymes and artificial substrate(s) for them, identification by enzyme immunoassay of one single cell, analyzing of different liquid or air samples. [0017] These advantages promise excellent opportunities for implementation of this device and its versions in medical diagnostics, industry, environmental science, and biodefense. SUMMARY OF THE INVENTION [0018] This invention constitutes a device consisting of a micro-channel plate, supportive structural elements, filter, and a filter holder, which is substituted by a pure agar block or nutrient media block during method performance. The device is intended for rapid detection and/or identification of microorganisms by providing biochemical enzymological or enzyme immunoassay reactions in a very small volume of the micro-channel. Even one single cell trapped in a micro-channel could be detected or/and identified in a time range of several minutes to tens of minutes. [0019] Microorganisms are trapped by filtration on the surface of filtrating material, e.g., a filter in the present embodiment, and contained in long (diameter/length=l/10- 1/100), cylindrical, parallel, micro-channels, also known as micro-channels or microchannels, that are open from both sides, and attached to a filtration material from one side. A micro-channel plate houses a multiplicity of micro-channels (possible diameter of each channel=l-30 um, length 100-lOOOum. and number of micro-channels per one cm =100,000- 1,000,000). After filtration is completed, the device is untwisted and the filter holder removed and substituted by a pure agarose block. The pure agarose block (agar) is filled by a biochemical indicator (artificial substrate(s) for enzyme(s) — chromogenic or fluorogenic, depends on method) reagent in advance. Molecules of the artificial substrate and solvent fill all micro-channels and natural enzyme(s) of a live cell or enzyme attached to the cell surface through an antigen — antibody reaction (enzyme immunoassay for single cell), which starts a transformation of colorless artificial substrate molecules into colored or fluorescent molecules. These molecules collect in a very small volume of micro-channel containing a cell. The extremely small volume of a micro-channel (1/25 Millionth part of milliliter) allows it to collect a detectable concentration of colored or fluorescent substance in ,a very short time. The volume of one^micro-channel is so small (only 40,000 cubic micrometers in the case of micro- channel's dimensions: diameter 10 micrometers and length 500 micrometers) that one single cell trapped in a micro-channel corresponds to a concentration of 25 million cells per milliliter of sample. Micro-channels containing cells and concentrated colored (violet, blue, dark blue, black or other — depends on used chromogenic substrate) molecules look like colored round spots on a bright background under a regular light microscope. Micro-channels containing cells and concentrated fluorescent molecules look like bright (blue, green, red — depends on used fluorogenic substrates) round spots on a dark background under a fluorescent microscope. The number of colored or fluorescent spots (dots) corresponds to the number of live cells in the initial filtrated sample or the number of special dangerous or pathogenic cells identified by enzyme immunoassay. A simple observation and count of colored or fluorescent dots on the surface of the micro- channel plate allows for rapid and reliable detection and/or identification and enumeration of cells in concentration of as little as one single cell per sample. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incoiporated in and form a part of this specification, illustrated embodiments of the invention and together with the description, serve to explain the principles of the invention. [0020] FIG. 1. shows the general structure and shape of the micro-channel glass plate in accordance with one aspect of the present disclosure. [0021] FIG. 2. explains mam parts and general structure of the device for sampling, detection, and/or identification of microorganisms in accordance with one aspect of the present disclosure. [0022] FIG. 3 shows differences between micro-channels filled by colored molecules (left) and fluorescent molecules (right) in accordance with one aspect of the present disclosure. [0023] FIG. 4 shows inner structure of the assembled device for sampling by filtration and placing cells present in a sample in micro-channels on the surface of filtration material in accordance with one aspect of the present disclosure. [0024] FIG. 5 shows components of the same device as on FIG. 4 unassembled in accordance with one aspect of the present disclosure. [0025] FIG. 6 demonstrates the real working model of the device shown on FIG. 4 and FIG. 5 in accordance with one aspect of the present disclosure. [0026] FIG. 7 demonstrates how, after filtration is done, the upper part of the device is removed from the filtrating lower funnel and the filter holder is removed and substituted by pure agar block, in accordance with one aspect of the present disclosure. [0027] FIG. 8 shows unassembled components of the device shown on FIG. 7 in accordance with one aspect of the present disclosure. [0028] FIG. 9 demonstrate the position of the device during filtration process: device with funnel for liquid samples (left), device with syringe for adding antibody-enzyme conjugate (center), device during air (bioaerosols) filtration (right), in accordance with one aspect of the present disclosure. [0029] FIG. 10 shows the general structure of the device transformed for depositing of magnetic particles with bioparticles (cells, viruses, biomolecules) adjusted to the surface of magnetic particle through Ab-Ag interaction in micro-channels in accordance with one aspect of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION [0030] This invention constitutes a device for rapid detection and/or identification of cells in concentrations up to a single cell present in an investigated sample without preliminary growth. This goal is reached by filtration of the investigated sample through a device consisting of a micro-channel plate, filter, and a holder for the filter, which is substituted during the procedure by an agarose block filled with reagents. The device is surrounded by a supportive frame. During filtration microorganisms pass through micro-channels and are trapped on the surface of the filter. After filtration is complete, micro-channels are filled by reagents from the agarose block, and a reaction between cellular enzymes and artificial substrate(s) begins. The product of these reactions (colored or fluorescent molecules) fills the volume of the micro-channel rapidly because of its extremely small inner volume. The product will quickly reach a detectable concentration seen under light or fluorescent microscope. Micro-channels containing cells look like colored or fluorescent dots easily distinct from empty micro-channels. [0031] The important part of the described device is a micro-channel plate (FIG. 1). The micro-channel plate is made of a glass disc that can contain thousands or millions of tiny, precisely manufactured channels (pores). Deviation of the micro-channels' size and volume usually doesn't exceed 1%. The channels are typically 1- 20 microns in diameter and have a length 10-100 times longer than their diameter. The volume of one channel could be from 1000 to 300,000 urn3. A micro-channel plate may contain 100,000-1,000,000 channels per cm2. The distance between channels is approximately 1-2 urn. The diameter of a plate could be varied. Usually it is 25-47 mm. Thickness of plates ranges from 0.2 to 5 mm (usually 0.5 mm). [0032] One single cell trapped in the channel (length-500 urn, diameter of channel- 10 jam, volume-40,000 jam 3) corresponds to a concentration of 25 million cells per ml. Thus, a detectable concentration of analytical (colored or fluorescent) substances from one cell will be reached in the same time that 25 million analogous cells are capable of producing. This time is several minutes or several tens of minutes, depending on the cells present and the method used. Another example of the effectiveness of micro volumes: concentration of a fluorescent substance which is visible by the naked eye (4-Methylumbelliferone from 4-Methylumbelliferyl acetate) is reached by one cell (Bacillus megatherium) in micro-channel (volume=40,000 jam 3) in two minutes. The same cell in one milliliter (volume=1012, ujn3) will produce the same concentration after 95 years. [0033] The micro-channel plate is made from a special glass. It is resistant to the different solvents or glass cleaning solutions. The micro-channel plate is also physically durable. The micro-channel can be made from black non-fluorescent glass or from non-colored glass. [0034] Micro-channels exhibit extremely strong capillary forces because of the very small diameter of the channels (capillaries). In fact, its capillary forces are powerful enough to raise a column of water up to a hundred meters. Even highly viscous liquids like Glycerol can easily fill the channels. Therefore, micro-channels will fill with liquids from an agarose block attached to the underside of the plate in a matter of seconds. [0035] Some micro-channel plates are not suitable enough for microbiological purposes of the invented device because their channels are manufactured under a special angle to the surface of the plate. This does not give the plate reader a chance to observe the entire inner volume of the micro-channel under a microscope. The optimal angle for viewing the channels is exactly 90°. Also, the production process of plates needs to be changed in order to produce colorless plates to use for color reactions. FIG. 3 shows the differences between micro channels filled by colored molecules (left) and fluorescent molecules (right). The pictures illustrate how both versions looks under light or fluorescent microscopes. [0036] The micro-channel plate shown on FIG. 1 has relatively big and short (small ratio of length/width) channels only for demonstration purposes. The real dimensions of the micro-channel plate used in this invented device are: diameter of the channel=10 urn, length of channel=500 um, number of micro-channels on one cm around 700,000, diameter of the plate 25 mm. It is possible to produce micro-channel plates with other parameters. The modern price of one micro-channel plate falls within a range of S50-S300. Each micro-channel plate can be used at least one hundred times for analytical puiposes in the invented device. [0037] The main parts of the device ready for filtration are shown in FIG. 2. The device consists of a micro-channel plate, filter, and a holder for the filter or agar block filled by artificial substrates. This structure is surrounded by a frame as shown in FIG. 4 (assembled) and FIG. 5 (taken apart): 1 — frame for plate, filter and filter holder done from plastic; 2 — rubber ring to prevent sudden damage of micro- channel plate and unlikable slots between frame and plate; 3 — micro-channel plate; 4 — filter for trapping cells; 5 — holder for filter able to pass liquids (liquid samples) or air (bioaerosols) done from plastic or glass particles; 6 — rubber ring for gently pressing holder for filter during filtration; 7 — plastic funnel for adjusting of the device to filtrating manifold (see FIG. 7 also). The upper part (1-5) stays together with the plastic funnel (7) by screw-in (frame (1) to funnel (7)) or by friction. The left side of Fig. 2 shows the position of the main components; micro channel plate, filter, filter holder, and agar block. In contrast, the right side of Fig. 2 shows a part of the structure at high magnification. The production of fluorescent or colored molecules from one cell trapped in micro-channel is shown in the channels. [0038] The procedure of filtrating is shown on the FIG. 9. The assembled device is attached to the manifold for filtrating. Negative pressure from the pump passes liquid or air samples through the micro-channel plate. Cells present in the sample are trapped in some of the micro-channels on the surface of the filter. [0039] When the filtrating process is complete and no more liquid is present inside the micro-channels, the device is removed from the manifold and untwisted. The plastic holder for the filter is removed, and the block with artificial substrate(s) is installed in its place (FIGS. 7 and 8). Immediately, all micro-channels fill with artificial substrate solution and a reaction takes place between enzymes of live cell or enzymes attached to the cellular surface through antibody (enzyme immunoassay). This process takes place in a significantly different (modified) device shown in FIGS. 7 and 8. As shown, FIGS. 7 and 8 demonstrate an assembled device containing: 1 - frame for plate, filter, and filter holder done from plastic; 2 — rubber ring to prevent sudden damage of micro-channel plate and unlikable slots between frame and plate; 3 — micro-channel plate, 4 - filter for trapping cells, 9 - agarose block filled by artificial substrates which in solution fill micro-channels; 10-- plate for agar block and attachment to frame 1. Plate 10 can be screwed up to frame 1 or just kept in place by friction. After needed changes are done, this modified device is placed in an incubator in order to create a detectable amount of colored or fluorescent molecules in micro-channels containing targeted microorganisms. Agar Block and Artificial Substrates [0040] The artificial substrates for different enzymes or enzymatic groups for the production of detectable concentrations of absorbent or fluorescent molecules are well-known. Artificial substrates are used for detection of enzymatic activities, live cell detection, and identification in enzyme immunoassay and ELISA. The main feature of artificial substrates is their ability to create colored or fluorescent molecules after transformation by enzymes. Many different artificial substrates are based on chromogenic molecules such as 2-Nitrophenol, 4-Nitrophenol, 5-Bromo-4-chloro- 3-Indoxol, 3-Inoxol, 5-Bromo-chloro 3-indoxol, 6-Chloro-3-indoxol, 5-Iodo-3-indoxol, N-Methylindoxol, 3,3'5,5'-Tetramethylibenzidine dihydrochloride, Tetrazolium salts, and others. Other artificial substrates are based on fluorescent molecules such as 4-Methylumbelliferone, 7-Amido-4-methylcoumarin, Fluorescein, Eosine and others. They cover a large spectrum of different enzymes such as Glycosidases, Esterases, Phosphatases, Peptidases, Sulfatases, Dehydrogenases, and special enzymes like Horseradish-Peroxidase, P-D-galactosidase, or a specific aminopeptidase. Some colored or fluorescent molecules produced from artificial substrates are collected inside the cell, while some of them go outside and are collected in the extracellular environment. Molecules collected inside the cell (Tetrazolium salts, 5-Iodo-3-indoxol, Fluorescein, and other) are important for Flow Cytometry and Epi-fluorescence because they color the cell body and make it more visible or/and detectable. This is a very small amount of molecules because it is restricted by the volume of cell. The collecting of these molecules inside a cell can cause cell death. Other group of molecules/substances has the ability to flow out of the cell during enzyme — substrate reactions (4-Methylumbelliferone, 7-Amido-4-methylcoumarin, 4-Nitrophenol and other). They do not cause cell death and therefore can collect for a long time and reach large concentrations. This group of artificial substrates is used for creating methods of detection and identification with the invented device because molecules collected in the micro-channel with the cell color them or make them fluorescent as shown in FIG. 3. The left side of FIG. 3 demonstrates the difference between a micro-channel filled by colored molecules as a result of a color reaction and micro-channels without cells. The circle below demonstrates colored micro-channel under a microscope. The right side of FIG. 3 demonstrates the differences between micro-channels filled by fluorescent molecules and micro-channels without cells. The circle below demonstrates micro-channels filled by fluorescent molecules under a microscope. [0041] Delivery of artificial substrates in micro-channels can be done with the help of an agarose cylindrical block filled by the needed artificial substrate (FIG. 2, FIG. 7 and FIG. 8). [0042] The advantage of the block filled with agarose gel (agar) is that dissolved substances (substrates) are not antagonistic to molecules-polymers of agar and therefore can easily go out and fill all micro-channels because of their strong capillary forces; the agar block can be squeezed in each direction and therefore easily fit to the surface of the filter without holes or slots. No one known artificial substrate does not react with agarose molecules. The agar block can be cut out from an agar layer or prepared by solidifying in special form. Agar is transparent to light and therefore can be used under a light microscope without removing. [0043] Other gels like gelatin, silica gels or polyacrylamide gel or even other earners of soluble substrates like filter paper can be used in some cases as well. Version of Device for Colored (Light Absorbent) Reactions [0044] The device used for color (light absorbent) reactions and molecules must have a colorless microchannel plate and colorless or white filter. Colorless micro-channel plate and filter are transparent to light whereas colored micro-channels are observed better with a light microscope in the light mode. Version for Fluorescent Reactions [0045] The black micro-channel plate and black filter must be used in this version in order to eliminate possible background fluorescence. Version for Particles (Coaled by Antibodies Non-Magnetic, Magnetic and Paramagnetic Microparticles) [0046] The invented device can be used for depositing in micro-channels not only cells but also particles coated by antibodies. These particles need to be significantly less than the diameter of the micro-channels. Particles coated by a specific monoclonal antibody (polypropylene, polycarbonate, magnetic and other) are broadly available on the diagnostic market. Coated particles are used for concentration of antigens (bacteria, viruses, proteins, and so on) on their surface by Ab-Ag interaction. Thus, particles coated by an antibody for an investigated virus will absorb viruses on their surface and after filtration through device, they will deposit in the micro-channels. Coated particles give opportunities to deposit in micro-channels small objects like viruses, proteins, and other biomolecules that could not be captured by regular filtration because of small size. After coated particles with separately attached viruses or biomolecules are trapped in the micro-channels, they can be identified by the enzyme immunoassay described above. [0047] Magnetic particles can be trapped in micro-channels by a magnetic field. In this case, the agar block shown in FIG. 7 must be substituted by a magnet. The magnet must be strong enough to pull magnetic particles from liquid to the bottom of the micro-channel as shown in FIG. 10. The ability to pull magnetic particles with antigens on the surface of filtration material from a liquid stream depends on the energy of the magnetic field, concentration of Iron, Cobalt and rare earth atoms, and/or size of particle and speed of stream. All these parameters can be easily adjusted for the specific needs of a sample in order to pull all magnetic particles from the stream and let nonmagnetic particles (organic and mineral particles, live and dead cells without needed antigen determinants and solvent molecules) flow out of the device flow chamber. [0048] This version of the device has an additional element: a lid with channels for input and output liquid loaded by magnetic particles with cells, viruses, or molecules attracted to their surface. FIG. 10 shows the structure of this modified device: 1 — frame for plate; 2 — rubber ring to prevent sudden damage of micro-channel plate and unlikable slots between frame and plate filter, and filter holder done from plastic; 3 — micro-channel plate; 4 — filter for trapping cells; 11 — Iron or rare earth (Neodymium and Samarium Cobalt, Neodymium Iron Boron alloys), magnet disk with the same size as agar block; 10 — plate for magnet or agar block; 12 — lid covers upper part of frame 1 and forming flow chamber; 8 — channels for input and output of liquid containing magnetic particles with antigens. FIG. 10 shows the process of depositing of magnetic particle on the bottom of micro-channel and it separating from non-magnetic particles. [0049] The principal scheme of magnet works is presented on the bottom of FIG. 10: magnetic and non-magnetic particles are easily separated in the magnetic field. Non-magnetic particles flow out the chamber, while magnetic particles with antigens are collected in micro-channels. The methods of antigens detection and/or identification are the same as for cells trapped by filtration discussed above. EXAMPLE 1 Detection of Microbial Contamination in Liquid Samples [0050] Many different liquid samples in food or pharmaceutical industry must be tested for the presence of bacteria or fungi. Approximately 100 ml of liquid sample, presumably containing microbes, is filtrated through the device shown in FIGS. 4, 5 and 6. The liquid is easily passed through a black filter (size of pores is 0.2 microns, cellulosic or nitrocellulosic) but cells are trapped at the bottom of the channels on the surface of the filter. After this device is untwisted and a porous disk is removed and substituted by an agar disk preliminarily filled with 4-Methylumbelhferyl phosphate and 4-Methylumbelliferyl acetate (0.1 ml of each with concentration 0.5 mg/ml). The mixture of these fluorogenic substrates guarantees that all live cells will be found because both substrates correspond to big groups of enzymes present in all live cells — Esterases and Phosphatases. Liquid from the agar block containing molecules of named fluorogenic substrates fills all micro-channels in several seconds. The device (with microbial cells in: some of the channels) is placed in an incubator (temperature 40-45° Celsius) for 20-30 minutes. After incubation the device is placed under a fluorescent microscope with excitation light around 300-380 nm and fluorescence around 420-480 nm. The micro-channels containing live cells can be easily differentiated by bright blue fluorescence in comparison with dark "empty" micro- channels. Even one single live cell in 100 ml can be reliably found in 30-40 minutes instead of 3-5 days by a conventional method of growth on Petri plates. EXAMPLE 2 Identification of Escherichia coli 0:157 in Samples [0051] A 100 ml sample of liquid is filtrated through the device using a white nitrocellulose filter and colorless micro-channel plate. About 2 ml of standard conjugated antibody for E. coli 0:157 antigens with Horseradish Peroxidase (HRP) are added to the device and slowly — part by part, in several minutes — filtrated through the device; the conjugate (Ab+HRP) is attached to the surface of E. coli 0:157 if the cells are present in some of the channels; After that, 50 ml of distilled water is filtrated through the device in order to wash out the rest of the conjugate. An agar block containing: solution of 3,3',5,5'-Tetramethylbenzidine is added to the device instead of porous holder for filter. Incubation is for 35-40 minutes at 40° C. After incubation, the device with filter is placed under a light microscope (multiplication =X100). Micro-channels containing E. coli 0:157 appear as blue dots. Other micro-channels appear as white dots. Even one cell in 100 ml could be found in less than one hour using this method and device. The regular procedure needs a preliminary growth period of at least 24-48 hours on Petri plates. Flow cytometry allows a microbiologist to find one cell colored by antibody+fluorochrom, practically immediately after it passes a laser beam in the detection zone; however, it needs many hours to pass 100 ml of sample through a 10 micron nozzle of detection zone. The price of Flow cytometers is around $100,000. PCR reaches the same results in about 3-4 hours and involves a complicated and expensive technique. In addition, the reliability of PCR analysis in mixtures of different species is not good enough. EXAMPLE 3 Detection and Identification by Coated Magnetic Particles [0052] Detection and identification of bacteria, viruses, and biomolecules in a sample with the help of magnetic particles consists of several stages. First stage: addition of magnetic particles coated by antibody to the sample presumably containing detected organisms or biomolecules. In this stage, the object is attached to the magnetic particle by Ab-Ag interaction. Second stage: liquid containing magnetic particles together with mixture of other particles passes through the device (FIG. 10). Magnetic particles are separated in micro-channels by magnetic field, and other particles flow out through output channel. Third stage antibody-enzyme conjugate passes through array of micro-channels and is attached to antigens trapped on magnet particles: bacteria, viruses, or biomolecules. Fourth stage: substitute magnet with transparent porous holder for filter and wash out the surplus of conjugate by distilled water: Fifth stage: remove porous holder for filter and add agar block containing artificial substrate to enzyme of conjugate. Sixth and seventh stages: incubate the device at 35-45° C. for 15-45 minutes (depends on conjugate and object), and enumerate colored or fluorescent (depends on version) micro-channels. Color or fluorescence in some micro-channels indicates presence of object attached to magnetic particle in the first stage. This procedure is generally common for all biological objects, such as cells, viruses, and biomolecules. Some objects can have such a small number of antigenic sites that attached enzymes (indicator molecules) are not able to reach a detectable concentration under a microscope (light or fluorescent). This drawback can be improved by use of smaller micro-channels and/or implementation of a more sophisticated source of excitation light (mini UV lasers) and photomultiplier (image intensifier) instead of visual detection. What is Claimed 1. An apparatus for detecting, identifying, or enumerating microorganism(s) or particle(s) with attached biomolecule(s), microorganism(s), or virus(es). the apparatus comprising: a device having a plurality of micro-channels wherein each of the plurality of micro-channels has a diameter of approximately 30 jam or less for detecting as few as one microorganism, or one particle with attached biomolecules(s), microorganism(s) or vims(es) per micro-channel: and a filtrating material for filtering the microorganism(s) or particle(s) with attached biomolecules(s), microorganism(s), or virus(es). 2. The apparatus according to claim 1, further comprising: a holder to hold the filtrating material against the device having a plurality of microchannels for trapping the microorganism(s) or particle(s) with attached biomolecule(s) or virus(es) in the micro-channels. 3. The apparatus according to claim 1, wherein the device having the plurality of micro-channels and the filtrating material are either black, colorless, or white. 4. The apparatus according to claim 2, wherein the holder for the filtrating material is substituted by a device with a magneticfield for depositing magnetic particle(s) coated with antibodies specific for biomolecule(s), microorganism(s), virus(es), or antigens that are attracted to the antibodies through an antibody-antigen interaction. 5. The apparatus according to claim 1, wherein the device having the plurality of micro-channels is use dfor performing color or fluorescent reactions, and wherein the device is a micro-channel glass plate with an angle between the micro-channels and the surface of the glass plate of approximately 90° 6. The apparatus according to claim 1, further comprising: an artificial substrate for detecting, identifying or enumerating microorganism(s) or particle(s) with attached biomolecule(s), microorgamsm(s), or virus(es), wherein the artificial substrate is selected from the group consisting of gels such as agarose, gelatin, polyacrylamide, or silica; paper; or porous material capable of keeping liquid with dissolved molecules inside for transfer to the micro-channels. T\|. j (new) The apparatus according to claim 1, wherein the plurality of micro-channels are open on both ends thereby enabling the plurality of micro-channels to be filled with liquid via capillary force. 8. (new) The apparatus according to claim 1, wherein the length of the micro-channels is approximately 1000 pm or less. 9 (new) The apparatus according to claim 1, wherein the volume of a micro-channel is approximately 300,000 um3 or less. 10. (new) The apparatus according to claim 1, wherein one microorganism per micro- channel provides a corresponding concentration of millions of cells per milliliter of sample. 11. (new) The apparatus according to claim 1, further comprising: a plurality of particles coated with an antibody (to which) biomolecule(s), microorganism(s), or virus(es) are specifically attached, wherein the particles together with the attached biomolecule(s), microorganism(s), or virus(es) are sufficiently large to be captured by the filtrating material, \|and wherein the biomolecule, microorganism, or virus by themselves may. not be large enough to be captured by the filtration material. 12. (new) The apparatus according to claim 1, further comprising: a lid coupled to the device having the plurality of micro-channels, the lid having \|an input and output in order to transport liquid loaded with! magnetic particle(s) witn attached biomolecule(s), microorganism(s), or virus(es); and ja device with a magnetic field] for [separating magnetic particle(s) into the plurality of micro-channels. 13. (new) A me'.hod of detecting, identifying, or enumerating microorganism(s), or particle(s) \| with attached biomolecule(s), microorganism(s), or virus(es), the method comprising the steps of: providing a device with a plurality of micro-channels; providing an] indicator \| to the micro-channels that rapidly reacts with the microorganism or the particle with attached biomolecule(s), microorganism(s), or virus(es) thereby creating a detectable concentration in the micro channel for as few as one microorganism or one particle with attached biomolecule(s), microorganism(s), or virus(es). 14. (new) The method according to claim 13, further comprising the steps of: providing a filtrating material; and filtering a sample through the filtrating material thereby trapping ^nicroorganism(s), or particle(s) with attached biomolecule(s), microorganism(s), or virus(es) in the micro-channels. 15. (new) The method according to claim 13, further comprising the step of: introducing liquid into the microchannels via capillary force). 16. (new) The method according to claim 13, further comprising the step of: detecting, identifying, or enumerating: i) dangerous or pathogenic microorganisms for medical diagnostics of human or veterinary medicine; ii) microbes for industrial microbiology; iii) contaminants of air, water, and soil, for environmental analysis; and/or iv) contagious or pathogenic microorganisms for epidemiology or biodefense. 17. (new) The method according to claim 13, wherein the indicator is one or more biochemical indicator reagent(s) selected from the group consisting of fluorogenic or chromogenic substrates; special enzymes and artificial substrate(s); enzyme immunoassays; and a conjugate with an artificial substrate. 18. (new) The me hod according to claim 13, further comprising the step of: enumerating micro-channel(s) having an appearance of colored or fluorescent dots] to determine a concentration of microorganism(s) in a sample based on the results of the enumeration step and on the volume of the sample. 19. (new) The method according to claim 13, wherein the (indicator is confined to a micro volume thereby maintaining a concentration level of indicator substance sufficient to provide detection. 20. (new) The me'hod according to claim 13, further comprising the steps of: adding to the sample, magnetic (particle(s) coated with an antibody foi) a microorganism, biomolecule, or virus that is sought; trapping the magnetic particle(s) in micro-channels by a magnetic fiel4 and identifying, enumerating, or detecting a microorganism by enzyme or enzyme immunoassay or iden'ifying, enumerating, or detecting a biomolecule by enzyme immunoassay. 21. (new) An apparatus for detecting,] identifying, or enumerating microorganism(s) or particle(s) with attached biomolecule(s) or virus(es), the apparatus comprising: a device having a plurality of micro-channels, wherein each of the plurality of micro-channels has a micro volume of approximately 300,000 urn3 or less, and wherein as few as \|one microorganism!, \|or ons particle with attached biomolecule(s), microorganism(s), or virusfes), per channel provides a (detectable color] or fluorescent (concentration with an indicator. 22. (new) A method for detecting,! identifying, or enumerating microorganism(s)j or particle(s) with attached biomolecule(s), microorganism(s), or virus(es)!, the method comprising the steps of: providing a device with a plurality of micro-channels that are open on both ends; trapping the microorganism(s) or particle(s) with attached biomolecule(s), micrborganism(s), or virus(es) in the micro-channels; and providing ja licuid that will fill the plurality of micro-channels via capillary action,. 23. (new) An apparatus for detecting] identifying, or enumerating microorganism(s)' or particle(s) with attached microorganism(s), virus(es), or biomolecule(s), the apparatus comprising: means for filtering a sample; means for trapping microorganism(s), or particle(s) with attached biomolecule(s), microorganism(s), or virus(es) in a micro-channel; means for adding an indicator to the micro-channel for detecting as few as one" microorganism or one particle with attached microorganism(s), virus(es), or biomolecules(s); and means for concentrating the indicator within the micro-channel to prevent dispersion such that a single cell can te detected without preliminary growth. 24. (new) A method for detecting,) identifying, or enumerating microorganism(sj or particle(s) coated with biomolecule(s), microorganism(s), or virus(es), the method comprising the steps of: providing a device with a plurality of microchannels; providing a sa:nple to be tested; and providing a volume of reagent to test the sample, wherein the volume of reagent required for testing is equivalent to the product of the quantity of the plurality of microchannels in the device times a micro-channel volume of approximately 300,000 um3 or less. 25. (new) A me'.hod for detecting,-identifying, or enumerating microorgamsm(s)\| or particle(s) coated with biomolecule(s), microorganism(s), or virus(es), the method comprising the steps of: providing a device with a plurality of channels for trapping the microorganism(s)( or particle(s) coated with biomolecule(s), microorganism(s), or virus(es); providing a sa nple to be tested in the device with the plurality of channels; and changing a co'responding concentration within a channel from zero cells per milliliter to at least five orders of magnitude of cells per milliliter by trapping as few as one cell in the channel.

Full Text

DEVICE FOR RAPID DETECTION AND IDENTIFICATION OF SINGLE MICROORGANISMS WITHOUT PRELIMINARY GROWTH
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] Modern microbiological diagnostics and analysis serves for detection, enumeration, and identification of microorganisms present in different samples. In the areas of medical diagnostics arid veterinary medicine, these organisms are pathogenic or dangerous microbes in human or animal blood, internal organs, skin, tissues, respiratory organs, and so on. In the area of industrial microbiology, microbes commonly pollute technological processes, materials, equipment, and finished products. In environmental analysis, there is often microbial contamination of water, indoor and outdoor air, and various surfaces. In epidemiology and biodefense - highly contagious pathogenic microorganisms from human body or environment.
[0002] Time, quality, arid sensitivity of microbiological analyses arc crucial for two reasons. First, tens of thousands of domestic microbiological laboratories spend several billions of dollars annually for products and processes quality control and prevention of contamination and spoilage in industry. These laboratories also spend money to provide diagnostic tests for humans, animals, plants, food, personal care products, soil, and environment. A quick, reliable, streamlined diagnostic test can save companies millions of dollars in the long run. Second, several thousands of people die even in such highly developed countries as the United States because of a delay in medical treatment caused by the length of time for diagnostics. Decreasing the time it takes to run these microbiological diagnostics tests will result in an increase of analysis reliability and sensitivity. Ultimately, this can save thousands of human lives worldwide.
[0003] The present invention relates to detection and identification of microorganisms without preliminary growth. In particular, the present invention relates to detection and identification of microorganisms without preliminary growth.
2. Description of the Related Art
[0004] Modern methods of detection, enumeration, and identification of microorganisms can be divided into two main parts 1) the methods and devices that need preliminary growth (enriching) to create a detectable amount of cells; 2) methods that don't need preliminary growth because they are capable of analyzing as little as a single cell.
[0005] The first group includes growth in solid or liquid regular or selective nutrient medias. It also includes several immunological methods. Examples are latex and hemagglutination, antibodies on magnetic particles, enzyme immunoassays like ELISA and Western Blot, and "dipstick" methods. The first group also includes chromatography of fatty acids, infrared Raman and FTIR spectroscopy, mass-spectrometry, and ATP-, bio-, and chemiluminescence. These methods require hundreds to millions of pure cells for detection of a certain microorganism, and, therefore, long (many hours or days) preliminary growth.
[0006] The second group of methods and devices does not need preliminary growth because they are capable of detecting and/or identifying even a single cell. These methods and devices consist of a group of nucleic acid methods like PCR and its various modifications, Epi-fluorescent methods (fluorogenic substrate methods, immunofluorescence), and a group of Flow Cytometry methods.
[0007] In addition to other drawbacks, methods of detection and/or identification of cells without preliminary growth usually need very expensive and sophisticated equipment and the work of high-level professionals. For example, devices used for PCR include the expensive thennocycler and sophisticated fluorometer. PCR is also used only for identification purposes. Enumeration of initial contamination is not reliable with PCR usage. PCR is very sensitive to organisms which can contaminate the test itself.
[0008] Epi-fluorescence usually needs only a fluorescent microscope to detect a single cell marked by a fluorescent dye or fluorescent antibody (Ab+fluorochrom). However, the amount of fluorescent substance present is restricted by volume of the cell body or cell surface. The small size of the object (single microorganism) makes the detection of one cell very difficult, especially with large background fluorescence usually present in a majority of the samples. Substances that flow out of a cell or enzyme immunoassay of a single cell are impossible with Epi- fluorescent methods
because these indicator substances disperse in the surrounding space immediately but are not concentrated in a small volume like proposed by the current invention.
[0009] Flow Cytometry is based on a very complicated opto-electronics system. A flow cytometer consists of a sophisticated optical block, a block of electronics, a complicated hydrodynamic system, and a high-speed computer. Prices for different types of flow cytometers range from $50,000 to $140,000. Flow cytometers can analyze one single cell during its flow through channels with diameters of 10 microns each. This size of the channel is so narrow that it needs 17 hours to pass 100 ml of liquid through it, even if speed of flow is 20 meters per second. Therefore, flow cytometers are currently used in hematology because of large (5-6 million/ml) and more or less stable concentrations of blood cells. Also, flow cytometers are very effective as sorters of cell mixtures in cytology. Usage of flow cytometry in microbiology is not easy. In microbiology these instruments are not often used because microorganisms can create clusters with other particles and can be confused with natural particles or dead cells. If the concentration of cells in a sample is very small, the time of analysis goes up dramatically. Therefore, preliminary concentration or even enriching is needed for Flow Cytometry microbiological applications. Microbes are also much more diverse by size and shape than blood cells and, thus, mistakes occur very often.
[0010] It is known, and currently used in practice, that dividing a sample into small volumes helps to detect cell concentration faster. This effect depends on reaching a detectable concentration in a small volume faster than in a large volume. U.S. Pat. No. 5,716,798 describes the method for rapid detection of microorganisms in a container divided on a plurality of discrete zones, each of which can be separately monitored for microbial presence by reaching detectable cell concentrations after preliminary growth in some zones. This method gives time-saving of 10% to 40% in comparison with other methods. U.S. Pat. No. 5,770,440 is based on the same effect. The present invention differs from these patents because of the analysis of a single cell. No time-consuming preliminary growth or nutrient media are necessaiy.
[0011] U.S. Pat. No. 4,959,301 is based on dividing a sample with viable biological entities into micro-droplets and detecting entities by growth or by biochemical reactions of a single entity within a droplet. This method can indicate a single cell in less than 30 minutes in some variants. Nevertheless, it is technologically
complicated. Micro-droplets are produced with different volumes and require statistical analysis for calculating results. This method could be reproduced only in a laboratory by highly professional personnel with use of sophisticated and expensive equipment.
[0012] The proposed device has significant advantages in comparison with known methods:
[0013] It is capable of detecting and/or identifying by colored or fluorescent enzyme or enzyme-immunoassay as little as one single cell trapped in a micro-channel (one cell in one micro-channel corresponds to a concentration of 25 million cells per ml). Thus, no preliminary growth is needed, and a detectable concentration is reached in several minutes.
[0014] The price of the device and analysis is ten times less than Flow Cytometry or PCR (only a regular fluorescent or light microscope is needed for this diagnostic device). Also, the amount of reagent needed is substantially less than with the use of regular 96-well plates. As a result, the analysis is simple and cost- effective.
[0015] The device is simple to use and involves performing only regular filtration with just a few manipulations. Even non-specialists can easily adopt this device and procedure that is very important for their broad use.
[0016] Many different methods can be applied with the proposed device: detection of live cells by fluorogenic or chromogenic substrate(s), differentiation by special enzymes and artificial substrate(s) for them, identification by enzyme immunoassay of one single cell, analyzing of different liquid or air samples.
[0017] These advantages promise excellent opportunities for implementation of this device and its versions in medical diagnostics, industry, environmental science, and biodefense.
SUMMARY OF THE INVENTION
[0018] This invention constitutes a device consisting of a micro-channel plate, supportive structural elements, filter, and a filter holder, which is substituted by a pure agar block or nutrient media block during method performance. The device is intended for rapid detection and/or identification of microorganisms by providing biochemical
enzymological or enzyme immunoassay reactions in a very small volume of the micro-channel. Even one single cell trapped in a micro-channel could be detected or/and identified in a time range of several minutes to tens of minutes.
[0019] Microorganisms are trapped by filtration on the surface of filtrating material, e.g., a filter in the present embodiment, and contained in long (diameter/length=l/10- 1/100), cylindrical, parallel, micro-channels, also known as micro-channels or microchannels, that are open from both sides, and attached to a filtration material from one side. A micro-channel plate houses a multiplicity of micro-channels (possible diameter of each channel=l-30 um, length 100-lOOOum. and number of micro-channels per one cm =100,000- 1,000,000). After filtration is completed, the device is untwisted and the filter holder removed and substituted by a pure agarose block. The pure agarose block (agar) is filled by a biochemical indicator (artificial substrate(s) for enzyme(s) — chromogenic or fluorogenic, depends on method) reagent in advance. Molecules of the artificial substrate and solvent fill all micro-channels and natural enzyme(s) of a live cell or enzyme attached to the cell surface through an antigen — antibody reaction (enzyme immunoassay for single cell), which starts a transformation of colorless artificial substrate molecules into colored or fluorescent molecules. These molecules collect in a very small volume of micro-channel containing a cell. The extremely small volume of a micro-channel (1/25 Millionth part of milliliter) allows it to collect a detectable concentration of colored or fluorescent substance in ,a very short time. The volume of one^micro-channel is so small (only 40,000 cubic micrometers in the case of micro- channel's dimensions: diameter 10 micrometers and length 500 micrometers) that one single cell trapped in a micro-channel corresponds to a concentration of 25 million cells per milliliter of sample. Micro-channels containing cells and concentrated colored (violet, blue, dark blue, black or other — depends on used chromogenic substrate) molecules look like colored round spots on a bright background under a regular light microscope. Micro-channels containing cells and concentrated fluorescent molecules look like bright (blue, green, red — depends on used fluorogenic substrates) round spots on a dark background under a fluorescent microscope. The number of colored or fluorescent spots (dots) corresponds to the number of live cells in the initial filtrated sample or the number of special dangerous or pathogenic cells identified by enzyme immunoassay. A simple observation and count of colored or fluorescent dots on the surface of the micro-
channel plate allows for rapid and reliable detection and/or identification and enumeration of cells in concentration of as little as one single cell per sample.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incoiporated in and form a part of this specification, illustrated embodiments of the invention and together with the description, serve to explain the principles of the invention.
[0020] FIG. 1. shows the general structure and shape of the micro-channel glass plate in accordance with one aspect of the present disclosure.
[0021] FIG. 2. explains mam parts and general structure of the device for sampling, detection, and/or identification of microorganisms in accordance with one aspect of the present disclosure.
[0022] FIG. 3 shows differences between micro-channels filled by colored molecules (left) and fluorescent molecules (right) in accordance with one aspect of the present disclosure.
[0023] FIG. 4 shows inner structure of the assembled device for sampling by filtration and placing cells present in a sample in micro-channels on the surface of filtration material in accordance with one aspect of the present disclosure.
[0024] FIG. 5 shows components of the same device as on FIG. 4 unassembled in accordance with one aspect of the present disclosure.
[0025] FIG. 6 demonstrates the real working model of the device shown on FIG. 4 and FIG. 5 in accordance with one aspect of the present disclosure.
[0026] FIG. 7 demonstrates how, after filtration is done, the upper part of the device is removed from the filtrating lower funnel and the filter holder is removed and substituted by pure agar block, in accordance with one aspect of the present disclosure.
[0027] FIG. 8 shows unassembled components of the device shown on FIG. 7 in accordance with one aspect of the present disclosure.
[0028] FIG. 9 demonstrate the position of the device during filtration process: device with funnel for liquid samples (left), device with syringe for adding antibody-enzyme conjugate (center), device during air (bioaerosols) filtration (right), in accordance with one aspect of the present disclosure.
[0029] FIG. 10 shows the general structure of the device transformed for depositing of magnetic particles with bioparticles (cells, viruses, biomolecules) adjusted to the surface of magnetic particle through Ab-Ag interaction in micro-channels in accordance with one aspect of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0030] This invention constitutes a device for rapid detection and/or identification of cells in concentrations up to a single cell present in an investigated sample without preliminary growth. This goal is reached by filtration of the investigated sample through a device consisting of a micro-channel plate, filter, and a holder for the filter, which is substituted during the procedure by an agarose block filled with reagents. The device is surrounded by a supportive frame. During filtration microorganisms pass through micro-channels and are trapped on the surface of the filter. After filtration is complete, micro-channels are filled by reagents from the agarose block, and a reaction between cellular enzymes and artificial substrate(s) begins. The product of these reactions (colored or fluorescent molecules) fills the volume of the micro-channel rapidly because of its extremely small inner volume. The product will quickly reach a detectable concentration seen under light or fluorescent microscope. Micro-channels containing cells look like colored or fluorescent dots easily distinct from empty micro-channels.
[0031] The important part of the described device is a micro-channel plate (FIG. 1). The micro-channel plate is made of a glass disc that can contain thousands or millions of tiny, precisely manufactured channels (pores). Deviation of the micro-channels' size and volume usually doesn't exceed 1%. The channels are typically 1- 20 microns in diameter and have a length 10-100 times longer than their diameter. The volume of one channel could be from 1000 to 300,000 urn3. A micro-channel plate may contain 100,000-1,000,000 channels per cm2. The distance between channels is approximately 1-2 urn. The diameter of a plate could be varied. Usually it is 25-47 mm. Thickness of plates ranges from 0.2 to 5 mm (usually 0.5 mm).
[0032] One single cell trapped in the channel (length-500 urn, diameter of channel- 10 jam, volume-40,000 jam 3) corresponds to a concentration of 25 million cells per ml. Thus, a detectable concentration of analytical (colored or fluorescent) substances from one cell will be reached in the same time that 25 million analogous cells are capable of producing. This time is several minutes or several tens of minutes, depending on the cells present and the method used. Another example of the effectiveness of micro volumes: concentration of a fluorescent substance which is visible by the naked eye (4-Methylumbelliferone from 4-Methylumbelliferyl acetate) is reached by one cell (Bacillus megatherium) in micro-channel (volume=40,000 jam 3) in two minutes. The same cell in one milliliter (volume=1012, ujn3) will produce the same concentration after 95 years.
[0033] The micro-channel plate is made from a special glass. It is resistant to the different solvents or glass cleaning solutions. The micro-channel plate is also physically durable. The micro-channel can be made from black non-fluorescent glass or from non-colored glass.
[0034] Micro-channels exhibit extremely strong capillary forces because of the very small diameter of the channels (capillaries). In fact, its capillary forces are powerful enough to raise a column of water up to a hundred meters. Even highly viscous liquids like Glycerol can easily fill the channels. Therefore, micro-channels will fill with liquids from an agarose block attached to the underside of the plate in a matter of seconds.
[0035] Some micro-channel plates are not suitable enough for microbiological purposes of the invented device because their channels are manufactured under a special angle to the surface of the plate. This does not give the plate reader a chance to observe the entire inner volume of the micro-channel under a microscope. The optimal angle for viewing the channels is exactly 90°. Also, the production process of plates needs to be changed in order to produce colorless plates to use for color reactions. FIG. 3 shows the differences between micro channels filled by colored molecules (left) and fluorescent molecules (right). The pictures illustrate how both versions looks under light or fluorescent microscopes.
[0036] The micro-channel plate shown on FIG. 1 has relatively big and short
(small ratio of length/width) channels only for demonstration purposes. The real
dimensions of the micro-channel plate used in this invented device are: diameter of the channel=10 urn, length of channel=500 um, number of micro-channels on one cm around 700,000, diameter of the plate 25 mm. It is possible to produce micro-channel plates with other parameters. The modern price of one micro-channel plate falls within a range of S50-S300. Each micro-channel plate can be used at least one hundred times for analytical puiposes in the invented device.
[0037] The main parts of the device ready for filtration are shown in FIG. 2. The device consists of a micro-channel plate, filter, and a holder for the filter or agar block filled by artificial substrates. This structure is surrounded by a frame as shown in FIG. 4 (assembled) and FIG. 5 (taken apart): 1 — frame for plate, filter and filter holder done from plastic; 2 — rubber ring to prevent sudden damage of micro- channel plate and unlikable slots between frame and plate; 3 — micro-channel plate; 4 — filter for trapping cells; 5 — holder for filter able to pass liquids (liquid samples) or air (bioaerosols) done from plastic or glass particles; 6 — rubber ring for gently pressing holder for filter during filtration; 7 — plastic funnel for adjusting of the device to filtrating manifold (see FIG. 7 also). The upper part (1-5) stays together with the plastic funnel (7) by screw-in (frame (1) to funnel (7)) or by friction. The left side of Fig. 2 shows the position of the main components; micro channel plate, filter, filter holder, and agar block. In contrast, the right side of Fig. 2 shows a part of the structure at high magnification. The production of fluorescent or colored molecules from one cell trapped in micro-channel is shown in the channels.
[0038] The procedure of filtrating is shown on the FIG. 9. The assembled device is attached to the manifold for filtrating. Negative pressure from the pump passes liquid or air samples through the micro-channel plate. Cells present in the sample are trapped in some of the micro-channels on the surface of the filter.
[0039] When the filtrating process is complete and no more liquid is present inside the micro-channels, the device is removed from the manifold and untwisted. The plastic holder for the filter is removed, and the block with artificial substrate(s) is installed in its place (FIGS. 7 and 8). Immediately, all micro-channels fill with artificial substrate solution and a reaction takes place between enzymes of live cell or enzymes attached to the cellular surface through antibody (enzyme immunoassay). This process takes place in a significantly different (modified) device shown in FIGS. 7 and 8. As shown, FIGS. 7 and 8 demonstrate an assembled device containing: 1 - frame for plate,
filter, and filter holder done from plastic; 2 — rubber ring to prevent sudden damage of micro-channel plate and unlikable slots between frame and plate; 3 — micro-channel plate, 4 - filter for trapping cells, 9 - agarose block filled by artificial substrates which in solution fill micro-channels; 10-- plate for agar block and attachment to frame 1. Plate 10 can be screwed up to frame 1 or just kept in place by friction. After needed changes are done, this modified device is placed in an incubator in order to create a detectable amount of colored or fluorescent molecules in micro-channels containing targeted microorganisms.
Agar Block and Artificial Substrates
[0040] The artificial substrates for different enzymes or enzymatic groups for the production of detectable concentrations of absorbent or fluorescent molecules are well-known. Artificial substrates are used for detection of enzymatic activities, live cell detection, and identification in enzyme immunoassay and ELISA. The main feature of artificial substrates is their ability to create colored or fluorescent molecules after transformation by enzymes. Many different artificial substrates are based on chromogenic molecules such as 2-Nitrophenol, 4-Nitrophenol, 5-Bromo-4-chloro- 3-Indoxol, 3-Inoxol, 5-Bromo-chloro 3-indoxol, 6-Chloro-3-indoxol, 5-Iodo-3-indoxol, N-Methylindoxol, 3,3'5,5'-Tetramethylibenzidine dihydrochloride, Tetrazolium salts, and others. Other artificial substrates are based on fluorescent molecules such as 4-Methylumbelliferone, 7-Amido-4-methylcoumarin, Fluorescein, Eosine and others. They cover a large spectrum of different enzymes such as Glycosidases, Esterases, Phosphatases, Peptidases, Sulfatases, Dehydrogenases, and special enzymes like Horseradish-Peroxidase, P-D-galactosidase, or a specific aminopeptidase. Some colored or fluorescent molecules produced from artificial substrates are collected inside the cell, while some of them go outside and are collected in the extracellular environment. Molecules collected inside the cell (Tetrazolium salts, 5-Iodo-3-indoxol, Fluorescein, and other) are important for Flow Cytometry and Epi-fluorescence because they color the cell body and make it more visible or/and detectable. This is a very small amount of molecules because it is restricted by the volume of cell. The collecting of these molecules inside a cell can cause cell death. Other group of molecules/substances has the ability to flow out of the cell during enzyme — substrate reactions (4-Methylumbelliferone, 7-Amido-4-methylcoumarin, 4-Nitrophenol and other). They do not cause cell death and therefore can collect for a long time and reach
large concentrations. This group of artificial substrates is used for creating methods of detection and identification with the invented device because molecules collected in the micro-channel with the cell color them or make them fluorescent as shown in FIG. 3. The left side of FIG. 3 demonstrates the difference between a micro-channel filled by colored molecules as a result of a color reaction and micro-channels without cells. The circle below demonstrates colored micro-channel under a microscope. The right side of FIG. 3 demonstrates the differences between micro-channels filled by fluorescent molecules and micro-channels without cells. The circle below demonstrates micro-channels filled by fluorescent molecules under a microscope.
[0041] Delivery of artificial substrates in micro-channels can be done with the help of an agarose cylindrical block filled by the needed artificial substrate (FIG. 2, FIG. 7 and FIG. 8).
[0042] The advantage of the block filled with agarose gel (agar) is that dissolved substances (substrates) are not antagonistic to molecules-polymers of agar and therefore can easily go out and fill all micro-channels because of their strong capillary forces; the agar block can be squeezed in each direction and therefore easily fit to the surface of the filter without holes or slots. No one known artificial substrate does not react with agarose molecules. The agar block can be cut out from an agar layer or prepared by solidifying in special form. Agar is transparent to light and therefore can be used under a light microscope without removing.
[0043] Other gels like gelatin, silica gels or polyacrylamide gel or even other earners of soluble substrates like filter paper can be used in some cases as well.
Version of Device for Colored (Light Absorbent) Reactions
[0044] The device used for color (light absorbent) reactions and molecules must have a colorless microchannel plate and colorless or white filter. Colorless micro-channel plate and filter are transparent to light whereas colored micro-channels are observed better with a light microscope in the light mode.
Version for Fluorescent Reactions
[0045] The black micro-channel plate and black filter must be used in this version in order to eliminate possible background fluorescence.
Version for Particles (Coaled by Antibodies Non-Magnetic, Magnetic and Paramagnetic Microparticles)
[0046] The invented device can be used for depositing in micro-channels not only cells but also particles coated by antibodies. These particles need to be significantly less than the diameter of the micro-channels. Particles coated by a specific monoclonal antibody (polypropylene, polycarbonate, magnetic and other) are broadly available on the diagnostic market. Coated particles are used for concentration of antigens (bacteria, viruses, proteins, and so on) on their surface by Ab-Ag interaction. Thus, particles coated by an antibody for an investigated virus will absorb viruses on their surface and after filtration through device, they will deposit in the micro-channels. Coated particles give opportunities to deposit in micro-channels small objects like viruses, proteins, and other biomolecules that could not be captured by regular filtration because of small size. After coated particles with separately attached viruses or biomolecules are trapped in the micro-channels, they can be identified by the enzyme immunoassay described above.
[0047] Magnetic particles can be trapped in micro-channels by a magnetic field. In this case, the agar block shown in FIG. 7 must be substituted by a magnet. The magnet must be strong enough to pull magnetic particles from liquid to the bottom of the micro-channel as shown in FIG. 10. The ability to pull magnetic particles with antigens on the surface of filtration material from a liquid stream depends on the energy of the magnetic field, concentration of Iron, Cobalt and rare earth atoms, and/or size of particle and speed of stream. All these parameters can be easily adjusted for the specific needs of a sample in order to pull all magnetic particles from the stream and let nonmagnetic particles (organic and mineral particles, live and dead cells without needed antigen determinants and solvent molecules) flow out of the device flow chamber.
[0048] This version of the device has an additional element: a lid with channels for input and output liquid loaded by magnetic particles with cells, viruses, or molecules attracted to their surface. FIG. 10 shows the structure of this modified device: 1 — frame for plate; 2 — rubber ring to prevent sudden damage of micro-channel plate and unlikable slots between frame and plate filter, and filter holder done from plastic; 3 — micro-channel plate; 4 — filter for trapping cells; 11 — Iron or rare earth (Neodymium and Samarium Cobalt, Neodymium Iron Boron alloys), magnet disk with the same size as agar block; 10 — plate for magnet or agar block; 12 — lid covers
upper part of frame 1 and forming flow chamber; 8 — channels for input and output of liquid containing magnetic particles with antigens. FIG. 10 shows the process of depositing of magnetic particle on the bottom of micro-channel and it separating from non-magnetic particles.
[0049] The principal scheme of magnet works is presented on the bottom of FIG. 10: magnetic and non-magnetic particles are easily separated in the magnetic field. Non-magnetic particles flow out the chamber, while magnetic particles with antigens are collected in micro-channels. The methods of antigens detection and/or identification are the same as for cells trapped by filtration discussed above.
EXAMPLE 1 Detection of Microbial Contamination in Liquid Samples
[0050] Many different liquid samples in food or pharmaceutical industry must be tested for the presence of bacteria or fungi. Approximately 100 ml of liquid sample, presumably containing microbes, is filtrated through the device shown in FIGS. 4, 5 and 6. The liquid is easily passed through a black filter (size of pores is 0.2 microns, cellulosic or nitrocellulosic) but cells are trapped at the bottom of the channels on the surface of the filter. After this device is untwisted and a porous disk is removed and substituted by an agar disk preliminarily filled with 4-Methylumbelhferyl phosphate and 4-Methylumbelliferyl acetate (0.1 ml of each with concentration 0.5 mg/ml). The mixture of these fluorogenic substrates guarantees that all live cells will be found because both substrates correspond to big groups of enzymes present in all live cells — Esterases and Phosphatases. Liquid from the agar block containing molecules of named fluorogenic substrates fills all micro-channels in several seconds. The device (with microbial cells in: some of the channels) is placed in an incubator (temperature 40-45° Celsius) for 20-30 minutes. After incubation the device is placed under a fluorescent microscope with excitation light around 300-380 nm and fluorescence around 420-480 nm. The micro-channels containing live cells can be easily differentiated by bright blue fluorescence in comparison with dark "empty" micro- channels. Even one single live cell in 100 ml can be reliably found in 30-40 minutes instead of 3-5 days by a conventional method of growth on Petri plates.
EXAMPLE 2
Identification of Escherichia coli 0:157 in Samples
[0051] A 100 ml sample of liquid is filtrated through the device using a white nitrocellulose filter and colorless micro-channel plate. About 2 ml of standard conjugated antibody for E. coli 0:157 antigens with Horseradish Peroxidase (HRP) are added to the device and slowly — part by part, in several minutes — filtrated through the device; the conjugate (Ab+HRP) is attached to the surface of E. coli 0:157 if the cells are present in some of the channels; After that, 50 ml of distilled water is filtrated through the device in order to wash out the rest of the conjugate. An agar block containing: solution of 3,3',5,5'-Tetramethylbenzidine is added to the device instead of porous holder for filter. Incubation is for 35-40 minutes at 40° C. After incubation, the device with filter is placed under a light microscope (multiplication =X100). Micro-channels containing E. coli 0:157 appear as blue dots. Other micro-channels appear as white dots. Even one cell in 100 ml could be found in less than one hour using this method and device. The regular procedure needs a preliminary growth period of at least 24-48 hours on Petri plates. Flow cytometry allows a microbiologist to find one cell colored by antibody+fluorochrom, practically immediately after it passes a laser beam in the detection zone; however, it needs many hours to pass 100 ml of sample through a 10 micron nozzle of detection zone. The price of Flow cytometers is around $100,000. PCR reaches the same results in about 3-4 hours and involves a complicated and expensive technique. In addition, the reliability of PCR analysis in mixtures of different species is not good enough.
EXAMPLE 3 Detection and Identification by Coated Magnetic Particles
[0052] Detection and identification of bacteria, viruses, and biomolecules in a sample with the help of magnetic particles consists of several stages. First stage: addition of magnetic particles coated by antibody to the sample presumably containing detected organisms or biomolecules. In this stage, the object is attached to the magnetic particle by Ab-Ag interaction. Second stage: liquid containing magnetic particles together with mixture of other particles passes through the device (FIG. 10). Magnetic particles are separated in micro-channels by magnetic field, and other particles flow out through output channel. Third stage antibody-enzyme conjugate passes through array of micro-channels and is attached to antigens trapped on magnet particles: bacteria,

viruses, or biomolecules. Fourth stage: substitute magnet with transparent porous holder for filter and wash out the surplus of conjugate by distilled water: Fifth stage: remove porous holder for filter and add agar block containing artificial substrate to enzyme of conjugate. Sixth and seventh stages: incubate the device at 35-45° C. for 15-45 minutes (depends on conjugate and object), and enumerate colored or fluorescent (depends on version) micro-channels. Color or fluorescence in some micro-channels indicates presence of object attached to magnetic particle in the first stage. This procedure is generally common for all biological objects, such as cells, viruses, and biomolecules. Some objects can have such a small number of antigenic sites that attached enzymes (indicator molecules) are not able to reach a detectable concentration under a microscope (light or fluorescent). This drawback can be improved by use of smaller micro-channels and/or implementation of a more sophisticated source of excitation light (mini UV lasers) and photomultiplier (image intensifier) instead of visual detection.

What is Claimed
1. An apparatus for detecting, identifying, or enumerating microorganism(s) or
particle(s) with attached biomolecule(s), microorganism(s), or virus(es). the apparatus
comprising:
a device having a plurality of micro-channels wherein each of the plurality of micro-channels has a diameter of approximately 30 jam or less for detecting as few as one microorganism, or one particle with attached biomolecules(s), microorganism(s) or vims(es) per micro-channel: and
a filtrating material for filtering the microorganism(s) or particle(s) with attached biomolecules(s), microorganism(s), or virus(es).
2. The apparatus according to claim 1, further comprising:
a holder to hold the filtrating material against the device having a plurality of microchannels for trapping the microorganism(s) or particle(s) with attached biomolecule(s) or virus(es) in the micro-channels.
3. The apparatus according to claim 1, wherein the device having the plurality
of micro-channels and the filtrating material are either black, colorless, or white.
4. The apparatus according to claim 2, wherein the holder for the filtrating
material is substituted by a device with a magneticfield for depositing magnetic
particle(s) coated with antibodies specific for biomolecule(s), microorganism(s),
virus(es), or antigens that are attracted to the antibodies through an antibody-antigen
interaction.
5. The apparatus according to claim 1, wherein the device having the plurality
of micro-channels is use dfor performing color or fluorescent reactions, and wherein
the device is a micro-channel glass plate with an angle between the micro-channels and
the surface of the glass plate of approximately 90°
6. The apparatus according to claim 1, further comprising:
an artificial substrate for detecting, identifying or enumerating microorganism(s) or particle(s) with attached biomolecule(s), microorgamsm(s), or
virus(es), wherein the artificial substrate is selected from the group consisting of gels such as agarose, gelatin, polyacrylamide, or silica; paper; or porous material capable of keeping liquid with dissolved molecules inside for transfer to the micro-channels.
T|. j (new) The apparatus according to claim 1, wherein the plurality of micro-channels are open on both ends thereby enabling the plurality of micro-channels to be filled with liquid via capillary force.
8. (new) The apparatus according to claim 1, wherein the length of the micro-channels is approximately 1000 pm or less.
9 (new) The apparatus according to claim 1, wherein the volume of a micro-channel is approximately 300,000 um3 or less.
10. (new) The apparatus according to claim 1, wherein one microorganism per micro-
channel provides a corresponding concentration of millions of cells per milliliter of sample.
11. (new) The apparatus according to claim 1, further comprising:
a plurality of particles coated with an antibody (to which) biomolecule(s), microorganism(s), or virus(es) are specifically attached, wherein the particles together with the attached biomolecule(s), microorganism(s), or virus(es) are sufficiently large to be captured by the filtrating material, |and wherein the biomolecule, microorganism, or virus by themselves may. not be large enough to be captured by the filtration material.
12. (new) The apparatus according to claim 1, further comprising:
a lid coupled to the device having the plurality of micro-channels, the lid having |an input and output in order to transport liquid loaded with! magnetic particle(s) witn attached biomolecule(s), microorganism(s), or virus(es); and
ja device with a magnetic field] for [separating magnetic particle(s) into the plurality of micro-channels.
13. (new) A me'.hod of detecting, identifying, or enumerating microorganism(s), or
particle(s) | with attached biomolecule(s), microorganism(s), or virus(es), the method comprising
the steps of:
providing a device with a plurality of micro-channels;
providing an] indicator | to the micro-channels that rapidly reacts with the microorganism or the particle with attached biomolecule(s), microorganism(s), or virus(es) thereby creating a detectable concentration in the micro channel for as few as one microorganism or one particle with attached biomolecule(s), microorganism(s), or virus(es).
14. (new) The method according to claim 13, further comprising the steps of:
providing a filtrating material; and
filtering a sample through the filtrating material thereby trapping ^nicroorganism(s), or particle(s) with attached biomolecule(s), microorganism(s), or virus(es) in the micro-channels.
15. (new) The method according to claim 13, further comprising the step of:
introducing liquid into the microchannels via capillary force).
16. (new) The method according to claim 13, further comprising the step of:
detecting, identifying, or enumerating: i) dangerous or pathogenic microorganisms for medical diagnostics of human or veterinary medicine; ii) microbes for industrial microbiology; iii) contaminants of air, water, and soil, for environmental analysis; and/or iv) contagious or pathogenic microorganisms for epidemiology or biodefense.
17. (new) The method according to claim 13, wherein the indicator is one or more
biochemical indicator reagent(s) selected from the group consisting of
fluorogenic or chromogenic substrates; special enzymes and artificial substrate(s); enzyme immunoassays; and a conjugate with an artificial substrate.
18. (new) The me hod according to claim 13, further comprising the step of:
enumerating micro-channel(s) having an appearance of colored or fluorescent dots] to
determine a concentration of microorganism(s) in a sample based on the results of the enumeration step and on the volume of the sample.
19. (new) The method according to claim 13, wherein the (indicator is confined to a micro
volume thereby maintaining a concentration level of indicator substance sufficient to provide
detection.
20. (new) The me'hod according to claim 13, further comprising the steps of:
adding to the sample, magnetic (particle(s) coated with an antibody foi) a microorganism,
biomolecule, or virus that is sought;
trapping the magnetic particle(s) in micro-channels by a magnetic fiel4 and
identifying, enumerating, or detecting a microorganism by enzyme or enzyme
immunoassay or iden'ifying, enumerating, or detecting a biomolecule by enzyme immunoassay.
21. (new) An apparatus for detecting,] identifying, or enumerating microorganism(s) or
particle(s) with attached biomolecule(s) or virus(es), the apparatus comprising:
a device having a plurality of micro-channels, wherein each of the plurality of micro-channels has a micro volume of approximately 300,000 urn3 or less, and wherein as few as |one microorganism!, |or ons particle with attached biomolecule(s), microorganism(s), or virusfes), per channel provides a (detectable color] or fluorescent (concentration with an indicator.
22. (new) A method for detecting,! identifying, or enumerating microorganism(s)j or
particle(s) with attached biomolecule(s), microorganism(s), or virus(es)!, the method comprising
the steps of:
providing a device with a plurality of micro-channels that are open on both ends; trapping the microorganism(s) or particle(s) with attached biomolecule(s), micrborganism(s), or virus(es) in the micro-channels; and
providing ja licuid that will fill the plurality of micro-channels via capillary action,.
23. (new) An apparatus for detecting] identifying, or enumerating microorganism(s)' or
particle(s) with attached microorganism(s), virus(es), or biomolecule(s), the apparatus
comprising:
means for filtering a sample;
means for trapping microorganism(s), or particle(s) with attached biomolecule(s), microorganism(s), or virus(es) in a micro-channel;
means for adding an indicator to the micro-channel for detecting as few as one" microorganism or one particle with attached microorganism(s), virus(es), or biomolecules(s); and
means for concentrating the indicator within the micro-channel to prevent dispersion such that a single cell can te detected without preliminary growth.
24. (new) A method for detecting,) identifying, or enumerating microorganism(sj or
particle(s) coated with biomolecule(s), microorganism(s), or virus(es), the method comprising
the steps of:
providing a device with a plurality of microchannels;
providing a sa:nple to be tested; and
providing a volume of reagent to test the sample, wherein the volume of reagent required for testing is equivalent to the product of the quantity of the plurality of microchannels in the device times a micro-channel volume of approximately 300,000 um3 or less.
25. (new) A me'.hod for detecting,-identifying, or enumerating microorgamsm(s)| or
particle(s) coated with biomolecule(s), microorganism(s), or virus(es), the method comprising
the steps of:
providing a device with a plurality of channels for trapping the microorganism(s)( or
particle(s) coated with biomolecule(s), microorganism(s), or virus(es);
providing a sa nple to be tested in the device with the plurality of channels; and changing a co'responding concentration within a channel from zero cells per milliliter to
at least five orders of magnitude of cells per milliliter by trapping as few as one cell in the
channel.

Documents:

8893-delnp-2007-Abstract-(19-12-2013).pdf

8893-delnp-2007-abstract.pdf

8893-delnp-2007-Claims-(19-12-2013).pdf

8893-delnp-2007-claims.pdf

8893-DELNP-2007-Correspondence Others-(04-04-2014).pdf

8893-delnp-2007-Correspondence Others-(19-12-2013).pdf

8893-delnp-2007-Correspondence-Others-(03-06-2013).pdf

8893-delnp-2007-correspondence-others.pdf

8893-delnp-2007-Description (Complete)-(19-12-2013).pdf

8893-delnp-2007-description (complete).pdf

8893-delnp-2007-drawings.pdf

8893-delnp-2007-form-1.pdf

8893-delnp-2007-Form-13-(19-12-2013).pdf

8893-DELNP-2007-Form-2-(04-04-2014).pdf

8893-delnp-2007-Form-2-(19-12-2013).pdf

8893-delnp-2007-form-2.pdf

8893-delnp-2007-Form-3-(03-06-2013).pdf

8893-delnp-2007-form-3.pdf

8893-delnp-2007-form-5.pdf

8893-delnp-2007-pct-101.pdf

8893-delnp-2007-pct-237.pdf

8893-delnp-2007-pct-304.pdf

8893-delnp-2007-Petition-137-(19-12-2013).pdf

Form-1 and Form-2.pdf

Form-13.pdf

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Patent Number

260656

Indian Patent Application Number

8893/DELNP/2007

PG Journal Number

20/2014

Publication Date

16-May-2014

Grant Date

15-May-2014

Date of Filing

19-Nov-2007

Name of Patentee

NANOLOGIX INC.

Applicant Address

843 N. MAIN STREET, HUBBARD, OHIO 44425, U.S.A.

Inventors:

#	Inventor's Name	Inventor's Address
1	GAZENKO SERGEY	1106 PARKSIDE LANE, MASON, OHIO 45040 USA.

PCT International Classification Number

G01N 33/554

PCT International Application Number

PCT/US2006/015321

PCT International Filing date

2006-04-20

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	11/109,857	2005-04-20	U.S.A.