WO2009143555A1

WO2009143555A1 - Concentration filtering and analysis of fluids

Info

Publication number: WO2009143555A1
Application number: PCT/AU2009/000607
Authority: WO
Inventors: Russell Edward Vincent; Danielle Leanne Baker; Christobel Margaret Ferguson
Original assignee: Ecowise Australia Pty Ltd
Priority date: 2008-05-27
Filing date: 2009-05-15
Publication date: 2009-12-03

Abstract

The present invention relates to devices, systems, methods and kits for the concentration of components within fluids. The present invention also relates to the analysis of said components within said fluids, including the detection, identification and quantification of said components.

Description

Methods and devices for fluid analyses

Technical Field

Background of the Invention

The analysis of fluids is an important and necessary task in many different contexts. For example, in relation to drinking water analysis, it is important to assess the levels of components present in drinking water, such as potentially pathogenic microorganisms, including for example protozoa, bacteria and viruses. Such assessment is important not only in terms of continuing routine quality control of drinking water, but also in terms of combating intentional introduction of a harmful, toxic or pathogenic component into drinking water, for example, for the purposes of bioterrorism. Other examples of the need for the analysis of fluids include, but are not limited to, the assessment of biological or chemical components in effluent, river systems, sea water or any other body of fluid for scientific, environmental, quality control or safety reasons.

The ability to accurately detect, identify and quantify low amounts of components in fluids firstly requires the concentration of such components within the fluids. This concentration process must concentrate into a fluid test sample a sufficiently high proportion of the components present in the fluid such that subsequent analysis of the components is able to be undertaken in a sufficiently sensitive and accurate manner. In addition, it is desirable that such concentration process be achieved in the minimum possible time so as to facilitate rapid analysis of components in fluids.

Furthermore, due to the physical location of fluids that must be analyzed, for example including but not limited to water catchments, river systems, sewerage treatment plants, sewerage outfalls, lakes or oceans, it is necessary to either transport fluids to an appropriate testing facility for analysis (which typically does not aid rapidity of analysis), or alternatively to have the capacity to partially or completely analyze fluids in situ. Partial analysis in situ may involve the concentration of components within fluids in situ, followed by transport of the concentrated sample to a testing facility. Complete analysis in situ may additionally involve the ability to test concentrated samples in situ, for example, by applying the concentrated sample to a biosensor. In either case, such analysis would require a portable device or system that can remotely, autonomously and rapidly concentrate components in fluids. Optionally, and for complete analysis in situ, such device or system should also have the capacity to analyze the concentrated fluids, for example using a biosensor, with either real time or logged data collection. The requirement for autonomy in executing these tasks means that a device that is self-cleaning would be highly advantageous.

The present invention provides devices, systems, methods and kits for the concentration of components within fluids. The present invention also relates to the analysis of said components within said fluids, including the detection, identification and quantification of said components. In one specific embodiment, the present invention provides a device or system that can autonomously, remotely and rapidly concentrate components within fluids in situ, with such concentrated fluids then being transported to a testing facility for analysis. In another embodiment, the present invention provides a device or system that can also analyze said components in situ and collect real time or logged data from said analysis.

Summary of the Invention

According to a first aspect of the present invention, there is provided a device for concentrating at least one component in a fluid in situ, wherein said device comprises a first filter and a second filter, wherein the fluid is passed through the first filter and the second filter.

According to a second aspect of the present invention, there is provided a device for detecting at least one component in a fluid in situ, wherein said device comprises:

(a) a first filter and a second filter; and

(b) a biosensor wherein the fluid is passed through the first filter and the second filter, thereby forming a concentrated test sample, and wherein the concentrated test sample is contacted with the biosensor, said biosensor detecting the at least one component.

The at least one component may be selected from the group comprising eukaryotic or prokaryotic organisms, cells, organelles or any combination, fraction or part thereof, plants, yeasts, algae, protozoa, bacteria, mycoplasma, viruses, prions, proteins, peptides, polypeptides, immunoglobulins, biotin, substrates, enzymes, receptors, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, lipids, nucleic acids, macromolecules or any other molecule or any combination, fraction or part thereof. The fluid may comprise any mixture, suspension, dispersion, solution or combination thereof.

The fluid may be selected from the group comprising water, including river water, drinking water, catchment water, seawater, artesian water and bore water, sewerage, effluent, fermentation broths, liquids or fractions thereof, cell lysates, cell culture supernatants, cell extracts, cell suspensions, protozoan cultures or lysates, bacterial cultures or lysates, viral cultures or lysates, plant extracts or fractions thereof. The device may further comprise a fluid system. The fluid system may provide a means for:

(a) delivering the fluid to the first filter;

(b) delivering the fluid from the first filter to the second filter; and

(c) delivering the concentrated test sample to the biosensor. The device may further comprise a data collection device. The data collection device may collect data in real time or as logged data.

The device may further comprise a power supply. The device may further comprise a self-cleaning module. The device may be fully automated. According to a third aspect of the present invention, there is provided a method for concentrating at least one component in a fluid in situ, wherein said method comprises passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid.

According to a fourth aspect of the present invention, there is provided a method for detecting at least one component in a fluid in situ, wherein said method comprises:

(a) passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid and forming a concentrated test sample; and

(b) contacting the concentrated test sample with a biosensor, thereby detecting the at least one component in the fluid. According to a fifth aspect of the present invention, there is provided a method for concentrating at least one component in a fluid in situ, wherein said method comprises passing the fluid through the device of the first aspect.

According to a sixth aspect of the present invention, there is provided a method for detecting at least one component in a fluid, wherein said method comprises passing the fluid through the device of the second aspect.

According to a seventh aspect of the present invention, there is provided a kit for concentrating at least one component in a fluid in situ, wherein said kit comprises a first filter and a second filter, wherein the fluid is passed through the first filter and the second filter.

According to an eighth aspect of the present invention, there is provided a kit for detecting at least one component in a fluid in situ, wherein said kit comprises:

(a) a first filter and a second filter; and

According to a ninth aspect of the present invention, there is provided a kit for concentrating at least one component in a fluid in situ, wherein said kit comprises the device of the first aspect. According to a tenth aspect of the present invention, there is provided a kit for detecting at least one component in a fluid in situ, wherein said kit comprises the device of the second aspect.

According to an eleventh aspect of the present invention, there is provided a device for concentrating at least one component in a fluid, wherein the device comprises

(a) a first reservoir; (b) a second reservoir;

(c) a first filter;

(d) a third reservoir; and

(e) a second filter, wherein the fluid is drawn from the first reservoir into the second reservoir and passed through the first filter, thereby forming a first concentration sample in the second reservoir; and wherein the first concentration sample is drawn from the second reservoir into the third reservoir and passed through the second filter, thereby forming a second concentration sample in the third reservoir.

According to a twelfth aspect of the present invention, there is provided a method for concentrating at least one component in a fluid, wherein the method comprises: drawing the fluid from a first reservoir into a second reservoir and passing it through a first filter, thereby forming a first concentration sample in the second reservoir; and drawing the first concentration sample from the second reservoir into a third reservoir and passing it through a second filter, thereby forming a second concentration sample in the third reservoir.

According to a thirteenth aspect of the present invention, there is provided a device for detecting at least one component in a fluid, wherein the device comprises:

(a) a first reservoir;

(b) a second reservoir; (c) a first filter;

(d) a third reservoir;

(e) a second filter; and

(f) a biosensor, wherein the fluid is drawn from the first reservoir into the second reservoir and passed through the first filter, thereby forming a first concentrated sample in the second reservoir; wherein the first concentrated sample is drawn from the second reservoir into the third reservoir and passed through the second filter, thereby forming a second concentrated sample in the third reservoir; and wherein the second concentrated sample is contacted with the biosensor, said biosensor detecting the at least one component.

According to a fourteenth aspect of the present invention, there is provided a method for detecting at least one component in a fluid, wherein the method comprises: drawing the fluid from a first reservoir into a second reservoir and passing it through a first filter, thereby forming a first concentration sample in the second reservoir; drawing the first concentration sample from the second reservoir into a third reservoir and passing it through a second filter, thereby forming a second concentration sample in the third reservoir; and contacting the second concentration sample with a biosensor, thereby detecting the at least one component in the fluid. Definitions

Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers or steps. As used herein the terms "protein" and "polypeptide" may be used interchangeably and include a polymer made up of amino acids linked together by peptide bonds, including fragments or analogues thereof. Although the terms "polypeptide" and "protein" may be used interchangeably herein, a "polypeptide" may also constitute either a portion of a full length "protein" or a complete full length "protein". As used herein the term "substantially" means the majority but not necessarily all.

As used herein, the term "in situ", when used in relation to detection of a component in a fluid, or in relation to analysis of a fluid, means that the detection or analysis is undertaken at, or very near to, the original site at which the fluid is sourced.

The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge. Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings:

Figure 1. Sample concentration module with biosensor installation. Figure 2. SCM power supply and control enclosures

Figure 3. SCM front panel.

Figure 4. RTMC system overview screen.

Figure 5. SCM power supply and enclosure.

Figure 6. SCM control enclosure, Figure 7. River pump.

Figure 8. Reservoir and pump set up.

Figure 9. Sample prefilter.

Figure 10. Stage 1 (50L) reservoir.

Figure 11. Stage 1 sensors and valves. Figure 12. Stage 2 (2L) reservoir.

Figure 13. Stage 2 pump.

Figure 14. Stage 2 filter.

Figure 15. Stage 2 backpressure valve.

Figure 16. Stage 2 backpressure sensor. Figure 17. Stage 2 sensor and valves.

Figure 18. Stage 3 (200ml) reservoir.

Figure 19. Stage 3 pump and filter.

Figure 20. Stage 3 backpressure valve.

Figure 21. Stage 3 sensor and valves. Figure 22. Tween reservoirs.

Figure 23. 200ml Tween valve.

Figure 24. Bulk cleaning reservoirs.

Figure 25. Cleaning solution valves.

Figure 26. Schematic diagram of a preferred embodiment of the device for concentrating at least one component within a fluid.

Figure 27. Schematic diagram of a preferred embodiment of the sample concentration module.

Figure 28. Sample concentration equipment set up. Detailed Description of the Invention

The present invention provides devices, systems, methods and kits for the concentration of components within fluids. The present invention also provides methods for the analysis of said components within said fluids, including the detection, identification and quantification of said components.

The inventors have designed, assembled and herein disclose a device that can rapidly concentrate the amount of one or more components in a fluid, and that can then accurately and rapidly detect, identify and quantify such components in fluids. Such analysis can be undertaken and data generated either in real time or as logged historical data. In addition, the portability of the device means that it has the capacity to partially or completely analyze fluids in situ. Partial analysis in situ may involve the concentration of components within fluids in situ, followed by transport of the concentrated sample to a testing facility. Complete analysis in situ may additionally involve testing the concentrated samples in situ, for example, by applying the concentrated sample to a biosensor. In either case, the device the subject of the present invention can remotely, autonomously (with the exception of periodic maintenance) and rapidly concentrate components in fluids in situ. Optionally, and for complete analysis in situ, the device may be used to analyze the concentrated fluids in situ, for example using a biosensor, with either real time or logged data collection. The device is also self-cleaning, thereby contributing to its ability to operate in a remote and autonomous manner in situ. This ability to operate in remote areas in an automated capacity without the need for continuous human intervention overcomes difficulties in transporting bulk fluids, the components in which have not been concentrated, from the field to an appropriate testing facility for analysis. Such transport not only requires human intervention and is costly, but it also typically greatly increases the time required to analyze the fluid. In one embodiment, the device concentrates one or more components within a fluid. This process concentrates the components present in the fluid into a concentrated test sample so that subsequent analysis of the components is able to be undertaken in a sufficiently sensitive and accurate manner. The process involves passing the fluid through a first filter, wherein the overall volume of the fluid is decreased whilst the components in the fluid are substantially retained. This is followed by passage of the fluid through a second filter, again involving decrease in the overall volume of the fluid and substantial retention of the components within the fluid. Once the concentrated test sample has been generated via these at least two filtration steps, the sample may then be contacted with a biosensor or other detection, identification and quantification device. Due to the increased concentration of the components within the sample, the sensitivity of the biosensor is able to accurately detect, identify and quantify low amounts of the component in the fluid. Fluids

The fluids that are subject to concentration, and optionally in addition, analysis, may comprise any mixture, suspension, dispersion, solution or combination thereof. The fluid may be selected from the group comprising water, including river water, drinking water, catchment water, seawater, artesian water and bore water, sewerage, effluent, fermentation broths, liquids or fractions thereof, cell lysates, cell culture supematants, cell extracts, cell suspensions, protozoan cultures or lysates, bacterial cultures or lysates, viral cultures or lysates, plant extracts or fractions thereof. The present invention contemplates concentrating and optionally analyzing fluids in situ, meaning that the concentration and optionally the analysis physically takes place at, or very near to, the original site at which the fluid is sourced. Accordingly, the present invention may involve concentration and optionally analysis of fluids in many different environments, for example, including but not limited to water catchments, river systems, sewerage treatment plants, sewerage outfalls, lakes or oceans.

In particular embodiments of the present invention, the fluid may be drinking water. In other embodiments of the present invention, the fluid may be sewerage or effluent.

Components The components in the fluids to be concentrated and optionally analyzed may be selected from the group comprising eukaryotic or prokaryotic organisms, cells, organelles or any combination, fraction or part thereof, plants, yeasts, algae, protozoa, bacteria, rickettsiae, mycoplasma, viruses, toxins, prions, proteins, peptides, polypeptides, immunoglobulins, biotin, substrates, enzymes, receptors, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, lipids, nucleic acids, macromolecules or any other molecule or any fraction, part, variant, mutant or combination thereof.

In particular embodiments of the present invention, the component may be a protozoan. The protozoan may be selected from the group comprising Sarcodina, including coccidia, Cryptosporidia, Toxoplasma, Babesia, and Plasmodium, Mastigophora, including trypanosomes, Histomonas and Trichomonas spp., Acanthamoeba, Babesia, Balantidium, Besnoitia, Chilomastix, Cochlosoma, Cryptobia, Cryptosporidium, Cystoisospora, Dientamoeba, Eimeria, Encephalitozoon, Endolimax, Entamoeba, Frenkelia, Giardia, including Giardia lamblia, Haemoproteus, Hammondia, Hartmannella, Hepatozoon, Hexamita, Histomonas, lodamoeba, Isospora, Klossiella, Leishmania, Leucocytozoon, Naegleria, Parahistomonas, Pentatrichomonas, Plasmodium, Sarcocystis, Theileria, Toxoplasma, Trichomonas, Tritrichomonas, Trypanosoma, Tyzzeria, Wenyonella spp., or any fraction, part, variant, mutant or combination thereof.

In other embodiments of the present invention, the component may be a bacterium. The bacterium may be selected from the group comprising Vibrio cholera, Clostridium botulinum, Salmonella typhi, Shigella dysenteriae, Escherichia coli, including Enterohaemorrhagic Escherichia coli, serotype 0157 and other verotoxin producing serotypes, Bacillus anthracis, Campylobacter, Legionella pneumophila, Listeria monocytogenes, Brucella abortus, Brucella melitensis, Brucella suis, Chlamydia psittaci, Francisella tularensis, Burkholderia mallei (Pseudomonas mallei), Burkholderia pseudomallei (Pseudomonas pseudomallei), Yersinia pestis, Clostridium perfringens, including epsilon toxin producing types, or any fraction, part, variant, mutant or combination thereof.

In still other embodiments of the invention, the component may be a virus. The virus may be selected from the group comprising Hepatitis A, poliovirus, Coxsackie virus, Chikungunya virus,

Congo-Crimean haemorrhagic fever virus, Dengue fever virus, Eastern equine encephalitis virus,

Ebola virus, Hantaan virus, Junin virus, Lassa fever virus, Lymphocytic choriomeningitis virus, Machupo virus, Marburg virus, Monkey pox virus, Rift Valley fever virus, Tick-borne encephalitis virus (Russian Spring-Summer encephalitis virus), Variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, White pox, Yellow fever virus, Japanese encephalitis virus, Kyasanur Forest virus, Louping ill virus, Murray Valley encephalitis virus, Omsk haemorrhagic fever virus, Oropouche virus, Powassan virus, Rocio virus, St Louis encephalitis virus, Hendra virus (Equine morbillivirus), South American haemorrhagic fever (Sabia, Flexal, Guanarito), pulmonary and renal syndrome-haemorrhagic fever viruses (Seoul, Dobrava, Puumala, Sin Nombre), Nipah virus, Human immunodeficiency virus (HIV), SARS Coronavirus or any fraction, part, variant, mutant or combination thereof.

In yet still other embodiments of the invention, the component may be a rickettsia. The rickettsia may be selected from the group comprising Coxiella burnetii, Bartonella quintana (Rochalimea quintana, Rickettsia quintana), Rickettsia prowazeki, Rickettsia rickettsii, or any fraction, part, variant, mutant or combination thereof.

In further embodiments of the present invention, the component may be a toxin. The toxin may be selected from the group comprising Botulinum toxins, Clostridium perfringens toxins, Conotoxin, Ricin, Saxitoxin, Shiga toxin, Staphylococcus aureus toxins, Tetrodotoxin, Verotoxin and shiga-like ribosome inactivating proteins, Microcystin (Cyanginosin), Aflatoxins, Abrin, Cholera toxin, Diacetoxyscirpenol toxin, T-2 toxin, HT-2 toxin, Modeccin toxin, Volkensin toxin, Viscum Album Lectin 1 (Viscumin), Tetanus toxin, Palytoxin, Trichothecenes or any fraction, part, variant, mutant or combination thereof. In yet further embodiments of the present invention, the component may be a fungi. The fungi may be selected from the group comprising Coccidioides immitis, Coccidioides posadasii, or any fraction, part, variant, mutant or combination thereof.

The component may be a genetically modified organism. The genetically modified organism may be modified from any one or more of the organisms disclosed herein, or any fraction, part, variant, mutant or combination thereof.

In particular embodiments of the present invention directed to counter terrorism, the component may be any organism or any fraction, part, variant, mutant or combination thereof that is capable of causing harm to any animal, for example, human beings, or domestic or agricultural animals, or to any plant, for example, agricultural crops.

Devices and system configuration

The present invention provides devices for concentrating at least one component in a fluid in situ, wherein said device comprises a first filter and a second filter, wherein the fluid is passed through the first filter and the second filter.

The present invention also provides devices for detecting at least one component in a fluid in situ, wherein said device comprises a first filter and a second filter, and a biosensor, wherein the fluid is passed through the first filter and the second filter, thereby forming a concentrated test sample, and wherein the concentrated test sample is contacted with the biosensor, said biosensor detecting the at least one component.

The device has been designed around a control and measurement module, a sample concentrating module and a power supply module. Various sensors are employed to monitor levels, pressures, temperatures and other parameters. Valves and pumps are used to control the flow of the fluid sample and other liquids (detergents and cleaning solutions, for example). The device is preferably housed in a suitable enclosure to allow for easy transport to an operational site. In an alternative arrangement, the control and measurement module, sample concentrating module and power supply module may be each housed in separate enclosures designed to be easily interfaced with the other modules. In either arrangement, operation of the device, once appropriately configured and initiated, is fully automatic. The device may further comprise a power supply. Referring to Figure 26, a device 1 is shown comprising a power supply 10, a control and measurement module 20 and a sample concentrating module 30 positioned in communication with fluid supply 40, which facilitates intake of sample fluid into the sample concentrating module. Although Figure 26 shows an embodiment where the device is located over a fluid supply, it will be appreciated that the device can be located proximate to a fluid supply site. For example, the device can be located on land near a river, where a hose, pump or other plumbing system can be employed to facilitate intake of fluid from the river into the sample concentrating module.

The power supply module is designed to provide DC power for the device, although the person skilled in the art will appreciate and understand that where the operating context of the device permits, AC power may also be used, with appropriate alterations to the power supply module as required. In one embodiment, a rechargeable battery or other suitable low voltage power source provides 24 VDC power for the device. It will be appreciated that voltages of 12V and 5V may be generated by a DC-DC convertor, if required. Preferably, separate fuses provide protection for any 24V, 12V and 5V circuit. The control and measurement module comprises an internal memory and includes embodied software to operate the various sample concentrating module elements and to control the sample concentrating module process. Data collected by the control and measurement module from the various sensors is preferably stored for future use in comma separated variable length text file format (CSV format). However, it will be appreciated that other data formats would be suitable. A graphical user interface (GUI) provides the operator with a simple to understand view of the current system operation and also recent process and calibration information. An example of such a GUI may be seen in Figure 4.

The operation of the device is entirely under the control of the measurement and control module. When initiated, the measurement and control module will begin to measure the sensor outputs, monitor the pump and valve status and collect the appropriate data into its internal memory at predetermined times. The program scan rate will depend on the minimum sensor reading interval, but is preferably of the order of 1 second. At every scan, all sensor parameters are read with the results being placed into a temporary memory location. Derived parameters, such as reservoir volumes, may be calculated from these sensor parameters and the results may also be placed into the temporary memory location. These results and parameters may be displayed in real time, or may show historic logged data at a particular point in the process.

The status of the device may be monitored based upon sensor and equipment signals indicating, for example, the existence of an error condition. These conditions may include loss of mains power to the battery charges, and sensor malfunctions for example. Once an error condition has been detected, the device may be automatically shut down to prevent damage to the equipment. Additionally, an alarm signal may be transmitted to an operator.

The device may further comprise a fluid system. The fluid system may provide a means for delivering the fluid to the first filter, delivering the fluid from the first filter to the second filter and delivering the concentrated test sample to the biosensor. Referring to Figure 27, an embodiment of the sample concentration module 30 is shown comprising a fluid intake 310 connected to the output of a fluid pump 300. The intake of the fluid pump 300 can be in free communication with, and can draw fluid from, desired sites such as water catchments, river systems, sewerage treatment plants, sewerage outfalls, lakes or oceans. As shown in Figure 27, fluid from the fluid intake is prefiltered by prefilter 320 to remove debris that may clog the sample concentrating module fluid paths.

5 Having passed through the prefilter 320, fluid is stored in a first reservoir 330, which preferably includes a level sensor 331 to monitor the fluid level therein. The first reservoir 330 may have a capacity in a range of from 1L to 1000L, 5L to 900L, 10L to 800L, 15L to 700L, 2OL to 600L, 25L to 500L, 3OL to 400L, 31 L to 380L, 32L to 360L, 33L to 340L, 34L to 320L, 35L to 300L, 36L to 280L, 37L to 260L, 38L to 240L₁ 39L to 220L, 4OL to 200L, 41L to 180L, 42L to 160L, 43L to 140L,

I₀ 44L to 120L, 45L to 100L, 46L to 8OL, 47L to 6OL, 48L to 55L or 49L to 52L. In a preferred embodiment, the first reservoir 330 is approximately 5OL in capacity.

Fluid flow from the first reservoir 330 may be controlled by a first valve 332, and associated solenoid, to a waste line 390 or drawn through a second valve 333, and associated solenoid, to a second reservoir 340. The second reservoir 340 may have a capacity in a range of from 10OmL to is 100L, 20OmL to 9OL, 30OmL to 8OL, 40OmL to 7OL, 50OmL to 6OL, 60OmL to 5OL, 70OmL to 4OL, 80OmL to 3OL, 90OmL to 2OL, 1 L to 10L, 1.1 L to 9L, 1.2L to 8L, 1.3L to 7L, 1.4L to 6L, 1.3L to 5L, 1.7L to 4L, 1.8L to 3L, 1.85L to 2.5L, 1.9L to 2.2L, 1.95L to 2.1L or 1.97L to 2.05L. In a preferred embodiment, the second reservoir 340 is approximately 2L in capacity.

The second reservoir 340 is used to hold the sample during the first stage of the

20 concentration cycle, and includes a first outlet connected to a pump 346. Pump 346 is positioned to take fluid from the second reservoir 340 and pass it through a first filter 347, the output of which feeds back into the second reservoir. A backpressure valve (not shown) is positioned after the first filter to apply a back pressure during the first stage concentration cycle. A sensor disposed between the first filter 347 and an inlet to the second reservoir 340 is used to monitor the back

2₅ pressure during the first stage of the concentration cycle. Additionally, a level sensor 341 fitted to the second reservoir allows fluid levels to be monitored therein. During this first stage concentration cycle, this first concentration sample may be concentrated to volume in a range of from 1OmL to 10L, 5OmL to 9L, 10OmL to 8L₁ 15OmL to 7L, 20OmL to 6L, 25OmL to 5L₁ 30OmL to 4L₁ 31OmL to 3.8L, 32OmL to 3.6L₁ 33OmL to 3.4L, 34OmL to 3.2L, 35OmL to 3L, 36OmL to 2.8L₁ so 37OmL to 2.6L₁ 38OmL to 2.4L₁ 39OmL to 2.2L, 40OmL to 2L₁41OmL to 1.8L₁42OmL to 1.6L, 43OmL to 1.4L, 44OmL to 1.2L, 45OmL to 1L, 46OmL to 80OmL₁ 47OmL to 60OmL, 48OmL to 55OmL or 49OmL to 52OmL. In some embodiments, the sample is preferably concentrated to a volume of 50OmL₁ and the second reservoir 340 is preferably kept full from the first reservoir via the second valve 333 until the fluid drawn from the first reservoir has been drained. A second outlet from the second reservoir 340 allows fluid to be discharged to a waste line 390 or to be drawn through to a third reservoir 350. Fluid flow from the second reservoir 340 may be controlled by a first valve 342, and associated solenoid, to the waste line 390 or by a second valve 343, and associated solenoid, to the third reservoir 350. The third reservoir 350 may have a 5 capacity in a range of from 1OmL to 10L, 2OmL to 9L, 3OmL to 8L, 4OmL to 7L, 5OmL to 6L, 6OmL to 5L, 7OmL to 4L, 8OmL to 3L, 9OmL to 2L, 10OmL to 1L, 11OmL to 90OmL, 12OmL to 80OmL, 13OmL to 70OmL, 14OmL to 60OmL, 13OmL to 50OmL, 17OmL to 40OmL, 18OmL to 30OmL, 185mL to 25OmL, 19OmL to 22OmL, 195mL to 21OmL or 197mL to 205mL In a preferred embodiment, the third reservoir 350 is approximately 20OmL in capacity.

I₀ The third reservoir 350 is used to hold the sample during the second stage of the concentration cycle, and includes a first outlet connected to a pump 356. Pump 356 is positioned to take fluid from the third reservoir 350 and pass it through a second filter 357, the output of which feeds back into the third reservoir 350. A backpressure valve (not shown) is positioned after the second filter 357 to apply a back pressure during the second stage concentration cycle. A sensor is disposed between the second filter and an inlet to the third reservoir is used to monitor the back pressure during the second stage of the concentration cycle. Additionally, a level sensor fitted to the third reservoir allows fluid levels to be monitored therein. During this second stage concentration cycle, this second concentration sample may be concentrated to volume in a range of from 0.5mL to 1L, 1mL to 90OmL, 1.5mL to 80OmL, 2mL to 70OmL, 2.5mL to 60OmL, 3mL to

20 50OmL, 3.5mL to 40OmL, 4mL to 30OmL, 4.5mL to 20OmL, 5mL to 10OmL, 5.5mL to 9OmL, 6mL to 8OmL, 6.5mL to 7OmL, 7mL to 6OmL, 7.5mL to 5OmL₁ 8mL to 4OmL, 8.5mL to 3OmL, 9mL to 2OmL, 9.2mL to 18mL, 9.4mL to 16mL, 9.6mL to 14mL or 9.8mL to 12mL. In some embodiments, the sample is preferably concentrated to a volume of 1OmL. The third reservoir 350 is preferably kept full from the second reservoir 340 via the second valve 343 until the fluid drawn from the second

2₅ reservoir 340 has been drained.

A second outlet from the third reservoir 350 allows the concentrated fluid (that is, the second concentration sample) to be passed to a sample outlet. In one embodiment, the sample outlet may pass the concentrated fluid sample to a holding container that can be transported ex situ to a testing facility. In another embodiment, the sample outlet may pass the concentrated fluid sample

30 directly to a biosensor for testing of the sample, for the detection, identification or quantification of one or more components within the concentrated fluid sample. Fluid flow from the third reservoir 350 may be controlled by a first valve 352, and associated solenoid, to the waste line 390 or by a second valve 353, and associated solenoid, to the sample outlet.

The device may further comprise a data collection device. The data collection device may

35 collect data in real time or as logged data. Typically the data collection device comprises a biosensor. In some embodiments, the data collection device permits real time transmission of data that is collected in situ from the field to a receiving station, whereupon appropriate action can be taken in response to the content of the data transmitted. For example, transmission of data in real time indicating detection of a pathogenic microorganism in a particular body of water, for example, a drinking water source, may allow for that water source to be isolated from drinking water supply.

The device may further comprise a self-cleaning module. To increase the accuracy of fluid analyses, it is desirable to flush the sample concentrating module 30 after each sample test. A plurality of valves mounted to the first reservoir may be used to control the introduction of at least one cleaning solution into the first reservoir. The at least one cleaning solution may be selected from the group comprising sodium hydroxide, hydrogen peroxide, phosphoric acid or other suitable liquids, or any combination, fraction or part thereof.

To purge the first reservoir 330, valve 332 is opened to allow transfer of any cleaning solution and waste from the first reservoir into waste line 390. While valve 332 is open for purging, valve 333 can be closed to reduce the possibility that waste located in the first reservoir 330 will be carried into the second reservoir 340. Additionally, it is desirable to flush the second and third reservoirs in a similar manner to that described above. Valves mounted to each of the second and third reservoirs may be used to control the introduction of a polysorbate surfactant or other suitable liquids into each reservoir.

The device may be fully automated. In one embodiment, automation of the device may be achieved by automation of the fluid system, wherein a combination of automated sensors, valves and pumps allow at least one component within a fluid to be concentrated within a fluid test sample without the need for human intervention. Such automation may be achieved, for example, using a control and measurement module comprising an internal memory and including embodied software to operate the various sample concentrating module elements and to control the sample concentrating module process, as herein disclosed. In addition, persons skilled in the art will understand that automation of the cleaning of the device may also be a factor contributing to the overall automated nature of the device, and therefore its ability to operate in situ without the need for human intervention.

It will also be appreciated that standard components such as pumps, valves and other standard forms of control system equipment can be used to implement the invention, though in some cases it would be desirable to use custom designed hardware depending on environmental aspects. Biosensors

In some embodiments of the present invention, the use of a biosensor may be employed for the detection, identification and quantification of components that have been concentrated in a fluid test sample. A biosensor is an analytical device which converts a biological response into an electrical signal, and which is used for the detection of an analyte that combines a biological component with a physicochemical detector component. Biosensors therefore typically consist of a biological element (being the "component" in the context of the present invention), a detector element (which typically operates in a physicochemical way, for example, using optical, piezoelectric electrochemical, thermometric, or magnetic signals), and a transducer which associates between the biological and detection elements.

Biosensors are well known to those of skill in the art, and the skilled artisan will therefore readily appreciate and understand that different biosensors may be employed as part of the devices, systems, methods and kits of the present invention depending on the particular application of the present invention. The particular biosensor used in performance of the present invention may depend on, for example, the component(s) in the fluid that is sought to be detected, identified or quantified.

The use of biosensors to detect, identify or quantify nucleic acids has traditionally involved the use of labeled cDNA or cRNA targets derived from the mRNA of an experimental sample which are hybridized to nucleic acid capture probes attached to a solid support. By monitoring the amount of label associated with each hybridized event, it is possible to infer the abundance of each mRNA species represented. Such biosensors may involve the use of real time polymerase chain reaction (PCR) machines. Similarly, in order to detect non-nucleic acid molecules including proteins, polypeptides, cells, or other whole organisms such as protozoa, bacteria and viruses, a capture probe attached to a solid support, such as a monoclonal antibody, may be used.

The person skilled in the art will know that several on-chip amplification strategies, including but not limited to rolling circle amplification, branched DNA technology, catalyzed reporter deposition, dendritic tags, enzymatic amplification, and chemical amplification are available as biosensing methods. Such amplification strategies may be coupled with electronic transduction methods.

Suitable biosensors for use with the present invention include, but are not limited to, the BIOSENS biosensor (Biosensor Applications Sweden AB), or the AMBRI ICS Biosensor (AMBRI Limited). Methods for concentrating at least one component in a fluid

The present invention provides methods for concentrating at least one component in a fluid in situ, wherein said method comprises passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid. Methods for increasing the s concentration of at least one component within a fluid are disclosed herein, for example, at

Example 2 and Figure 28.

Methods for detecting at least one component in a fluid

The present invention provides methods for detecting at least one component in a fluid ino situ, wherein said method comprises passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid and forming a concentrated test sample, and then contacting the concentrated test sample with a biosensor, thereby detecting the at least one component in the fluid.

The present invention also provides methods for detecting at least one component in a fluid,s wherein said method comprises passing the fluid through the device of the present invention.

Kits

The present invention also provides kits for separating, purifying, removing, enriching and/or concentrating a component from a mixture or suspension, wherein the kits facilitate the0 employment of the systems and methods of the invention. Typically, kits for carrying out a method of the invention contain all the necessary reagents to carry out the method. Typically, the kits of the invention will comprise one or more containers, containing for example, wash reagents, and/or other reagents capable of releasing a bound component from a polypeptide or fragment thereof.

In the context of the present invention, a compartmentalised kit includes any kit in whichs reagents are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. Such kits may also include a container which will accept a test0 sample, a container which contains the polymers used in the assay and containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and like).

Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods.

Methods and kits of the present invention find application in any circumstance in which it is₅ desirable to purify any component from any mixture. The present invention therefore provides kits for concentrating at least one component in a fluid in situ, wherein said kit comprises a first filter and a second filter, wherein the fluid is passed through the first filter and the second filter.

The present invention also provides kits for detecting at least one component in a fluid in situ,

5 wherein said kit comprises a first filter and a second filter, and a biosensor, wherein the fluid is passed through the first filter and the second filter, thereby forming a concentrated test sample, and wherein the concentrated test sample is contacted with the biosensor, said biosensor detecting the at least one component.

The present invention additionally provides kits for concentrating at least one component in a io fluid in situ, wherein said kit comprises the device as disclosed herein.

The present invention moreover provides kits for detecting at least one component in a fluid in situ, wherein said kit comprises the device as disclosed herein.

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

I₅

Examples

Example 1. Device for concentrating components in a fluid

20 The following example discloses one embodiment on the present invention, being a demonstration of how one or more components may be concentrated within a fluid sample. Although this example does not present in situ operation of a device encompassed by the present invention, the skilled artisan will readily appreciate and understand that the set up of the device as herein disclosed is suitable for in situ operation in the field.

2₅

1.1 Device overview

A Sample Concentration Unit (hereafter called "SCM") was designed around a Campbell Scientific CR1000 Control & Measurement Module. Various sensors were employed to monitor levels, pressures, temperatures and other parameters. Valves and pumps were used to control the 30 flow of the sample and other liquids (detergents and cleaning solutions). The SCM hardware was mounted on a purposed built stand and was designed to be easily interfaced with a biosensor. The CR1000 and control components were housed within an enclosure. A second enclosure was used to house the power supply components. Operation of the SCM, once initiated, was fully automatic.

Figure 1 illustrates an SCM overview set up for operation in conjunction with a biosensor. At 3₅ the top of the SCM are 3 x 2OL bulk storage vessels for process cleaning solutions. Below this (not visible) are the 5OL first stage reservoir and the tap water reservoir. Mounted on the front panel are 2L and 20OmL concentration reservoirs, 2L and 20OmL peristaltic pumps, 2L and 20OmL filters, Tween detergent storage vessels and various pinch valves for liquid control. A syringe pump and control box (above and beside the 2L peristaltic pump) are part of a biosensor and are not required for SCM stand alone operation. A large 500L tank can be used for storage of either ultra pure (uP) water or an unconcentrated sample, depending upon SCM configuration. All the other equipment shown in Figure 1, such as an auto sampler, fridge and computer/UPS and sample culture/sensor module (the two plastic cases beneath the SCM front panel) do not form part of the SCM and are not required for stand alone operation of the concentration process. In Figure 2 are shown a power supply and CR1000 control enclosures and a computer monitor. Figure 3 shows the SCM front panel.

The CR1000 contains a program specifically written to read/operate the various SCM components and control the SCM processes. A computer running special software is used to program, control and monitor the SCM operation. As data is collected it is stored for future use in a simple .CSV format. A graphical user interface (GUI) provides the operator with a simple to understand view of the current system operation and also recent process and calibration information.

The CR1000 control module was programmed to collect data at a predetermined rate. Two sorts of data can be generated - instantaneous (most recent or 'real time' data) and historical (or 'logged') data. The real time data can be used for display on the GUI software, while the logged data can be retrieved for later analysis.

The CR1000 was regularly "polled" to collect the data using a software package called LoggerNet. Monitoring, programming and data retrieval is set and controlled using LoggerNet. LoggerNet allowed the user to configure the frequency at which the CR1000 was polled and data was collected (both 'real time' and historical). LoggerNet also provided maintenance features (such as a Status screen) and may also be used for 'manual' data collection.

When the CR1000 is polled by LoggerNet the most recent instantaneous (or 'real time') values are collected in addition to any data that has been logged since the last poll. If data is 'missed' (for example, during an IT network outage) LoggerNet will collect the most recent instantaneous values and all logged data since the last data collection.

The Campbell Scientific CR1000 datalogger is capable of storing many months of data. Data is collected as long as LoggerNet maintains its configuration and is running on the polling computer. This data is raw and unprocessed and is stored in CSV format on the computer in a folder structure. The data may be retrieved manually or automatically for processing. In addition LoggerNet maintains a data cache which is used, amongst other things, for generating the SCM displays. The data cache is stored in binary format.

A second software package called Real Time Monitoring & Control (RTMC) was used to generate the 'screens' or displays which show the current (and some historical) data as it is collected. RTMC as used in this example has four screens that provide: (1) SCM operation overview, (2) real time data records, (3) SCM process history, and (4) auto calibration information.

Figure 4 shows an RTMC system overview screen.

1.2 System operation The operation of the SCM was entirely under the control of the Campbell Scientific CR1000

Measurement & Control Module. When the SCM was programmed, it began to measure (or "read") the sensor outputs, monitor the pump and valve status and collect (or 'log') the appropriate data into its internal memory at predetermined times.

A exemplary SCM program was operated as follows-: (1) The CR1000 program scan rate was 1 second;

(2) Every scan all sensor parameters are read with the result being placed into a temporary memory location;

(3) Derived parameters (i.e. reservoir volumes) were calculated from these sensor parameters and the results were placed into a temporary memory location; (4) Upon a valid Start code being entered into the software, the SCM began one of three processes (see below). These processes were automatic and continued until the process was complete or over ridden by the user.

(5) Data was logged throughout the various processes when determined by the CR1000 program. The SCM was predominantly powered from a 12/24Vdc battery backed power supply. Once powered up and initialised the SCM was capable of running three different processes-:

(1) Sample Concentration

(2) Post Sample Wash

(3) Daily Wash The CR1000 was programmed to run 24hrs/day with complete four Sample Concentration /

Post Sample Wash cycles and Daily Wash cycles being completed in that period.

Each of the SCM processes follows a step-by-step sequence of events, identified by a Sequence Number. A user may start or stop a process at any stage by entering the appropriate Sequence Number into the software system. An example of the SCM processes may be seen in Tables 1 - 4 below. 1.3 SCM function

1.3.1 SCM power supply enclosure

The SCM power supply enclosure as shown in Figure 5 housed all of the SCM DC power systems. Two RFI SME240-12-10 and SME240-24-5 power supply / battery chargers charged two banks of suitably wired 6V SLA batteries. A 12Vdc-5Vdc DC-DC converter provided 5Vdc power. Separate fuses provided protection for the 5Vdc circuit (for the relay multiplexer), 2 x 12Vdc circuits (for the ISCO sampler and SCM controller) and a 24Vdc circuit (for a river pump and solenoid valves).

1.3.2 SCM control enclosure

As shown in Figure 6, the SCM control enclosure contained the Campbell Scientific CR1000 Measurement & Control Module, the relay multiplexer board and the termination strips for the various sensors, valves, pumps and control circuits to operate the SCM hardware, The relay multiplexer was used to expand the number of control devices that the CR1000 can operate. The relays were identified from Relay 1 to Relay 32 and, along with the devices they operated, were shown on the software overview screen. The relays were configured for operation with either 5Vdc, 12Vdc, 24Vdc or contact closure outputs.

Relay 0, the relay for controlling the 24Vdc river pump, was not operated via the relay multiplexer.

1.3.3 Shurflo River Pump (Pump 3)

As shown in Figure 7, a Shurflo river pump was used to pump the sample from a river (or other suitable body of water) into the Stage 1 reservoir. This pump was controlled using Relay 0.

1.3.4 Tap Water Reservoir & Tap Water Pump (Pump 4)

As shown in Figure 8, this reservoir was used for the storage of tap water for either sample concentration or cleaning processes. The person skilled in the art will understand that tap water may be replaced with any water source when the device is used in the field in situ. In this example tap water pump resided inside the reservoir and was used to pump water into the Stage 1 reservoir

1.3.5 Sample Prefilter(s)

As shown in Figure 9, sample water from the river pump passed through these filter(s) before entering the Stage 1 reservoir. The 100um filter was used to remove debris that may clog the SCM fluid paths. An additional 10um filter was sometimes installed to provide additional filtering. 1.3.6 Stage 1 (50L) First Reservoir

As shown in Figure 10, the Stage 1 reservoir was used to hold the unconcentrated sample or diluted cleaning solutions during system operation.

1.3.7 Stage 1 Sensor & Valves

As shown in Figure 11, a Druck PTX1400 level sensor was fitted to the T' piece outlet at the bottom of the 5OL reservoir to measure the sample level. The signal from this was fed to the

CR1000 where a level-to-volume calculation was made for display purposes. The Sample (left) and Waste (right) solenoid valves were used to control the flow of liquid to either waste or to the Stage 2

(2L) reservoir.

1.3.8 Stage 2 (2L) Second Reservoir

As shown in Figure 12, this reservoir was used to hold the sample while it was being concentrated. The reservoir was kept full from the 5OL reservoir via the 5OL sample valve. As the sample was pumped out of the 2L reservoir, around the 2L filter and back into the reservoir it was reduced in volume. Additional sample was added from the 5OL reservoir and the process repeated until the entire sample was reduced to approximately 50OmL.

1.3.9 Stage 2 Pump (Pump 1)

As shown in Figure 13, this pump is a modified ISCO 3700 auto sampler pump which was mounted into a PVC enclosure. It was used to pump the sample from the 2L reservoir through the 2L filter. Custom control circuitry enabled this pump to be operated either forward or reverse direction at either full or 1/2 full speed.

1.3.10 Stage 2 First Filter

As shown in Figure 14, a Millipore cellulose or polyurethane filter was used for the first stage of the sample concentration process. This filter was held in a modified Millipore stand to enable easy replacement.

1.3.11 Stage 2 Backpressure Valve

As shown in Figure 15, this pinch valve was used to apply a back pressure to the 2L fluid path during the concentration and cleaning cycles. A manual back pressure can also be applied via the knob at the top of the 2L filter mounting bracket. 1.3.12 Stage 2 Backpressure Sensor

As shown in Figure 16, a Druck PTX1400 level sensor was fitted to the bottom of the 2L filter mount to measure the back pressure in the 2L concentration system. The signal from this was fed to the CR1000 for display purposes.

1.3.13 Stage 2 Sensor & Valves

As shown in Figure 17, a Druck PTX1400 level sensor was fitted to the 'T' piece outlet at the bottom of the 2L reservoir to measure the sample level. The signal from this was fed to the CR1000 where a level-to-volume calculation is made for display purposes. The Sample (left) and Waste (right) pinch valves were used to control the flow of liquid to either waste or to the Stage 3 (20OmL) reservoir.

1.3.14Stage 3 (20OmL) Third Reservoir

As shown in Figure 18, this reservoir was used to hold the sample while it was being concentrated for the second time. The reservoir was kept full from the 2L reservoir via the 2L sample valve. As the sample was pumped out of the 20OmL reservoir, around the 20OmL filter and back into the reservoir it was reduced in volume. Additional sample were added from the 2L reservoir and the process repeated until the entire sample was reduced to approximately 1OmL.

1.3.15Stage 3 Pump & Second Filter

As shown in Figure 19, this pump is a Masterflex peristaltic pump mounted into the SCM front panel. It was used to pump the sample from the 20OmL reservoir through the 20OmL filter. The

20OmL 'Pelicon' filter was mounted on a bracket in front of the Masterflex pump in such a way as to reduce the volume of the 20OmL fluid path. The pump may be operated either forward or reverse direction at either full or 2/3 full speed.

1.3.16 Stage 3 Backpressure Valve

As shown in Figure 20, this pinch valve was used to apply a back pressure to the 20OmL fluid path during the concentration and cleaning cycles.

1.3.17 Stage 3 Sensor & Valves

As shown in Figure 21 , a Honeywell 24PC pressure sensor was fitted to the bottom of the 20OmL reservoir to measure the sample level. The signal from this was fed via a conditioning circuit to the CR1000 where a level-to-volume calculation was made for display purposes. The Sample (left) and Waste (right) pinch valves were used to control the flow of liquid to either waste or to the SCM sample outlet.

1.3.18Tween Reservoirs

5 As shown in Figure 22, these two reservoirs held the 0.1% and 0.01% Tween solutions used during the sample concentration process. The 0.1% Tween was introduced into the 2L reservoir after the sample had been concentrated from 5OL to 50OmL. The 0.01% Tween was introduced into the 20OmL reservoir after the sample had been concentrated from 50OmL to 1OmL.

I₀ 1.3.19Tween Valves

As shown in Figure 23, these two pinch valves were used to control the flow of Tween from the reservoirs into the 2L and 20OmL reservoir under the control of the CR1000 module.

1.3.20Cleaning Solutions & Valves is As shown in Figure 24, these three containers were used to store bulk cleaning solutions used by the SCM during the post sample and daily washes. The solutions used in this example were Sodium Hydroxide (NaOH), Hydrogen Peroxide (H2O2) and Phosphoric Acid (H3PO4).

As shown in Figure 25, three pinch valves, mounted on a bracket around the top of the 5OL reservoir, were used to control the flow of solution into the reservoir during the cleaning processes. 20

1.4 Software

The operation of the SCM software suite, once loaded and correctly configured, was continuous and automatic. No user intervention was required.

The SCM operating program resides on the CR1000 datalogger and controls the system 2₅ operation. It is advantageous, though not necessary, for this program to be also stored on the polling computer, where it can be quickly retrieved in the event that SCM re-programming is required.

The automated process controlled by the SCM may be programmed in a step-by-step manner to allow the user to accurately control the concentration process. The step-by-step 30 automation programming also allows the user to collect data at individual steps of the process.

As described above, scheduled data collection is under the control of the LoggerNet software and may occur as follows-:

(1) Once initiated LoggerNet will poll the CR1000 and, using software 'pointers', will locate the last data point collected. It then retrieves all the subsequent logged data and the current 35 instantaneous readings; (2) The logged data is stored in CSV format in data files as per the LoggerNet Setup. Data may be appended (preferred) into the files or it may be over written. In the SCM setup all the logged data files should be appended;

(3) The logged data is also stored in the LoggerNet data cache along with the instantaneous data. This data, which is in binary format, is used by the display software. Storage in the data cache occurs automatically - it requires no special set up by the user to be carried out;

(4) RTMC updates the SCM displays every second. These pages are configured as per the current RTMC project file;

(5) Data is stored into the computer local hard drive and is stored in several tables with user definable file names.

Configuration and operation of the SCM software suite preferably occurs in the following order:

(1) Start LoggerNet;

(2) Configure LoggerNet communications (i.e. radio) paths; (3) Setup the SCM 'station' in LoggerNet;

(4) Test the SCM connections;

(5) Monitor the SCM performance; and

(6) Start & Monitor the SCM RTMC display project.

Example 2. Concentration of a fluid sample

The following example demonstrates one embodiment of a concentration process in accordance with the present invention. The apparatus set up is shown schematically in Figures 26 and 27. However, a particularly preferred embodiment of the concentration equipment set up is shown in Figure 28. It will be appreciated that the individual steps may be achieved using the system components disclosed herein, or by the inclusion of additional components to the device herein disclosed. Such components would be considered routine additions by those of skill in the art.

2.1 First Concentration Sample of 5OL to 50OmL

2.1.1 Fill the 2L reservoir with 2L of raw water. 2.1.2 Switch on the pump to 5L/min.

2.1.3 Run until the sample volume falls to 50OmL.

2.1.4 Refill reservoir up to 2L mark with raw water.

2.1.5 Complete this process until all 5OL of raw water has been added to the 2L reservoir.

2.1.6 Periodically discard filtrate as necessary. 2.1.7 Stop the pump when the level in the 2L reservoir reaches 30OmL.

2.1.8 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 2L reservoir.

2.1.9 Add 5mL of Tween 0.1% to the 50OmL of retentate sample. 2.1.10 Start pump at Vz speed in forward motion for 15 minutes with the permeate line blocked 2.1.11 Stop pump after 15 minutes

2.12.12 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 2L reservoir.

2.2 Second Concentration Sample of 50OmL to 1OmL 2.2.1 Fill the 50OmL reservoir with the 50OmL of retentate sample from the previous concentration step.

2.2.2 Switch on the pump to 600r/min.

2.2.3 Run until the retentate sample volume falls to 7mL.

2.2.4 Periodically discard filtrate as necessary. 2.2.5 Complete this process until all 5OL of raw water has been added to the 2L reservoir.

2.2.6 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 50OmL reservoir.

2.2.7 Add 1mL of Tween 0.01% to the 1OmL of retentate sample.

2.2.8 Start pump at 14 speed in forward motion for 15 minutes with the permeate line blocked 2.2.9 Stop pump after 15 minutes

2.2.10 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 50OmL reservoir.

2.3 Water rinse of 2L reservoir and associated pump and manifold between samples

2.3.1 Fill the 2L reservoir with 2L of tap water. 2.3.2 Switch on the pump to 5L/min.

2.3.3 Run until the retentate sample volume falls to 30OmL.

2.3.4 Periodically discard filtrate as necessary.

2.3.5 Reverse the pump so all retentate rinse held in the tubing and filter casing is returned to the 2L reservoir. 2.3.6 Discard the 50OmL retentate.

2.3.7 Repeat

2.3.8 Fill the 2L reservoir with 2L of tap water

2.3.9 Switch on the pump to 5L/min.

2.3.10 Start pump at Vz speed in forward motion for 15 minutes with the permeate line blocked 2.3.11 Stop pump after 15 minutes

2.3.12 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 2L reservoir.

2.3.13 Discard the retentate. 2.3.14 Repeat steps 2.3.1 to 2.3.7

2.4 Water rinse of 50OmL reservoir and associated pump and manifold between samples

2.4.1 Fill the 50OmL reservoir with 50OmL of tap water.

2.4.2 Switch on the pump to 5600r/min. 2.4.3 Run until the retentate sample volume falls to 5OmL.

2.4.4 Periodically discard filtrate as necessary.

2.4.5 Reverse the pump so all retentate rinse held in the tubing and filter casing is returned to the 50OmL reservoir.

2.4.6 Discard the 5OmL retentate. 2.4.7 Repeat

2.4.8 Fill the 50OmL reservoir with 50OmL of tap water

2.4.9 Switch on the pump to 600r/min.

2.4.10 Start pump at ¹/₂ speed in forward motion for 15 minutes with the permeate line blocked

2.4.11 Stop pump after 15 minutes 2.4.12 Reverse the pump so all retentate sample held in the tubing and filter casing is returned to the 50OmL reservoir.

2.4.13 Discard the retentate.

2.4.14 Repeat steps 2.4.1 to 2.4.7

2.5 Daily chemical clean of 2L reservoir and associated pump and manifold between samples

2.5.1 Fill the 2L reservoir with 2L of 0.1 N NaOH.

2.5.2 Start pump at ¹/₂ speed in forward motion for 15 minutes with the permeate line blocked.

2.5.3 Stop pump after 15 minutes

2.5.4 Reverse the pump so all retentate chemical held in the tubing and filter casing is returned to the 2L reservoir.

2.5.5 Reverse the pump so all retentate rinse held in the tubing and filter casing is returned to the 2L reservoir.

2.5.6 Discard the retentate.

2.5.7 Run the complete water rinse program in 2.3 2.5.8 Fill the 2L reservoir with 2L of 1 % H2O2.

2. 5.9 Repeat steps 2.5.2 to 2. 5.7

2. 5.10 Fill the 2L reservoir with 2L 1N H3PO4

2.5.11 Repeat steps 2.5.2 to 2.5.7 2.6 Daily chemical clean of 50OmL reservoir and associated pump and manifold between samples

2.6.1 Fill the 50OmL reservoir with 50OmL of 0.1 N NaOH.

2.6.2 Start pump at Vi speed in forward motion for 15 minutes with the permeate line blocked.

2.6.3 Stop pump after 15 minutes 2.6.4 Reverse the pump so all retentate chemical held in the tubing and filter casing is returned to the 50OmL reservoir.

2.6.5 Reverse the pump so all retentate rinse held in the tubing and filter casing is returned to the 50OmL reservoir.

2.6.6 Discard the retentate. 2.6.7 Run the complete water rinse program in 2.4

2.6.8 Fill the 50OmL reservoir with 50OmL of 1 % H2O2.

2.6.9 Repeat steps 2.6.2 to 2.6.7

2.6.10 Fill the 50OmL reservoir with 50OmL 1N H3PO4

2.6.11 Repeat steps 2.6.2 to 2.6.7 An example of the SCM step-by-step automation process may be seen in Tables 1 - 4 below, wherein:

Table 1 shows the background process data to initiate the SCM allowing the concentrating process described above in Example 2;

Table 2 demonstrates an example of the program sequence to run the sample concentration process described above in Example 2;

Table 3 shows an example of a post-sample wash process programmed to the SCM; and Table 4 demonstrates an example of the program sequence to run a daily wash process described above in Example 2.

O

Example 3. Concentration of bacterial components in a fluid sample

3.1 Experimental background

In this example, several tests were performed to determine the rate of recovery of Escherichia coli bacteria by a device encompassed by the present invention. In particular, Escherichia coli concentration efficacy was testing by measuring seed recovery, rinse cycles were tested for Escherichia coli removal efficacy, sanitising cycles were tested for Escherichia coli removal efficacy, and sanitising cycles were tested for chemical persistence and effect on subsequent E. coli recovery.

Various sample matrices were also tested to ascertain the likely sample processing times. Results of these experiments are shown in Table 5 and Table 6.

Table 5: Maximum concentration speed for concentration steps*

Εxcludes cleaning schedule Table 6: Expected process time for sample concentration module

3.2 Methods

Preliminary experiments were conducted with a range of sample matrices to determine possible bacterial recovery efficiencies.

Subsequent tests were performed on 1OL of tap water as a test matrix. All runs included a rinse cycle after the concentration cycle. Run details (time for run and rinses, system pressure) were recorded on the run sheet.

Runs 2, 5 and 32 were blanks serving as a negative control. Runs 3, 6 and 33 were blanks which were seeded with a BioBall after concentration, which served as a positive control testing the recovery of the analytical method. Runs 4, 26, 29, 30 and 31 were the concentration of a tap water sample seeded with an E. coli BioBall. 3.3 Results

Results of the preliminary experiments are shown in Table 7, with comments as indicated for each sample type tested and the observed recovery rate.

Table 7: Mean Recovery for concentration steps

Table 7: Mean Recovery for concentration steps (continued)

Overall Sample Concentration Module

77% of 45% 35% To validate in the laboratory.

Will be optimised with improvement in the 1^st concentration step.

The results of two sets of subsequent experiments using tap water as the sole matrix are shown in Tables 8 and 9.

In the first set of experiments (Table 8), the results for the three blank runs (2, 5 and 32) indicate there is minimal if any carry over of bacteria between samples. The method control runs (3,

6 and 33) indicate that the method of enumeration is sufficiently accurate for the analysis of these samples. The average overall sample recovery efficiency for these samples spiked with E. coli was 28.6% measured across five runs.

In the second set of experiments (Table 9), the results for the five blank runs (81, 83, 87, 90 and 92) demonstrated no carry over of these bacteria between samples. The method control runs

(79, 84, 88 and 93) again indicate that the method of enumeration is sufficiently accurate for the analysis of these samples. The average overall sample recovery efficiency for this second set of samples spiked with E. coli was 4.39%, measured across seven runs.

Table 8: First experiment results for concentration of bacterial components in a fluid sample

Seeding key: EC = E.coli Biobali of 10,000 cells

Example 4. Concentration of protozoan components in a fluid sample

4.1 Experimental background

In this example, several tests were performed to determine the rate of recovery of Cryptosporidium and Giardia by a device encompassed by the present invention. In particular, Cryptosporidium and Giardia concentration efficacy was testing by measuring seed recovery, rinse cycles were tested for Cryptosporidium and Giardia removal efficacy, sanitising cycles were tested for Cryptosporidium and Giardia removal efficacy, and sanitising cycles were tested for chemical persistence and effect on subsequent recovery.

4.2 Methods

All tests were performed on 1OL of tap water as a test matrix. All runs included a rinse cycle after the concentration cycle. Run details (time for run and rinses, system pressure) were recorded on the run sheet. Runs 9, 11, 13, 35, 37 and 39 were to test efficacy at concentrating Cryptosporidium and

Giardia, and to see if the post-concentration rinse cycle was able to clear the system of Cryptosporidium and Giardia. Runs 9, 11, 13, 35, 37 and 39 were the concentration of a sample seeded with EasySeed. The resultant concentrate was then seeded with ColorSeed before laboratory analysis. The EasySeed recovery show recovery efficacy, and the ColorSeed recovery served as a positive control (internal control) testing the recovery of the analytical method.

Runs 10, 12, 14, 36, 38 and 40 were blank, serving as negative controls. These samples were analysed for Cryptosporidium and Giardia, to see whether there was any carryover from the previous spiked runs.

4.3 Results

The results of two sets of experiments using tap water as the sole matrix are shown in Tables 10 and 11. The recovery efficiency of the inoculated Cryptosporidium and Giardia are shown in the % Easyseed column. All runs were also inoculated post concentration with an internal control of ColorSeed Cryptosporidium and Giardia. The recovery of these organisms indicates the efficiency of the enumeration method and these results are shown in the Internal Control columns.

In the first set of experiments (Table 10), the results for the five blank runs (10, 12, 14, 36, 38 and 40) indicate there is minimal (up to 3%) if any carry over of Cryptosporidium oocysts and up to 1% carryover of Giardia cysts between samples. The results of the internal controls indicate that the method of enumeration was sufficiently accurate for the analysis of these samples. The average overall sample recovery efficiency for samples spiked with Cryptosporidium was 25.9% and for Giardia was 25.2% measured across six runs.

Table 10: First experiment results for concentration of protozoan components in a fluid sample

Seeding key: ES = EasySeed, CS = ColorSeed In the second set of protozoan concentration experiments (Table 11), the results for the five blank runs (96, 98, 100, 102 and 104) demonstrated no carry over of Cryptosporidium oocysts or Giardia cysts between samples. The results of the internal controls indicate that the method of enumeration was sufficiently accurate for the analysis of these samples. The average overall sample recovery efficiency for samples spiked with Cryptosporidium was 2.33% and for Giardia was 4.67% measured across five runs.

Claims

1. A device for concentrating at least one component in a fluid in situ, comprising: a first filter; and a second filter, wherein the fluid is passed through the first filter and the second filter.

2. A device for detecting at least one component in a fluid in situ, comprising: (a) a first filter and a second filter; and

3. The device according to claim 1 or claim 2, wherein the at least one component is selected from the group comprising eukaryotic or prokaryotic organisms, cells, organelles, yeasts, algae, protozoa, bacteria, mycoplasma, viruses, prions, proteins, peptides, polypeptides, immunoglobulins, biotin, substrates, enzymes, receptors, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, lipids, nucleic acids, macromolecules or any other molecule or any combination, fraction or part thereof.

4. The device according to claim 1 or claim 2, wherein the fluid may comprise any mixture, suspension, dispersion, solution or combination thereof.

5. The device according to claim 1 or claim 2, wherein the fluid is selected from the group comprising water, including river water, drinking water, catchment water, seawater, artesian water and bore water, sewerage, effluent, fermentation broths, liquids, cell lysates, cell culture supernatants, cell extracts, cell suspensions, protozoan cultures or lysates, bacterial cultures or lysates, viral cultures or lysates, plant extracts or any combination, fraction or part thereof.

6. The device according to claim 2, further comprising a fluid system, operable to provide means for: (a) delivering the fluid to the first filter;

(b) delivering the fluid from the first filter to the second filter; and

(c) delivering the concentrated test sample to the biosensor.

7. The device according to claim 1 or claim 2, further comprising a data collection device.

8. The device according to claim 7, wherein the data collection device may collect data in real time or as logged data.

9. The device according to claim 7, further comprising a power supply.

10. The device according to claim 7, further comprising a self-cleaning module.

11. The device according to claim 1 or claim 2, wherein the concentrating at least one component in a fluid in situ, or detecting at least one component in a fluid in situ is automated.

12. A method for concentrating at least one component in a fluid in situ, comprising passing the fluid through the device according to claim 1.

13. A method for detecting at least one component in a fluid in situ, comprising passing the fluid through the device according to claim 2.

14. A kit for concentrating at least one component in a fluid in situ, comprising the device according to claim 1.

15. A kit for detecting at least one component in a fluid in situ, comprising the device according to claim 2.

16. A method for concentrating at least one component in a fluid in situ, comprising: passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid.

17. A method for detecting at least one component in a fluid in situ, comprising: (a) passing the fluid through at least two filters, thereby increasing the concentration of the at least one component in the fluid and forming a concentrated test sample; and

(b) contacting the concentrated test sample with a biosensor, thereby detecting the at least one component in the fluid.

18. A kit for concentrating at least one component in a fluid in situ, comprising: a first filter; and a second filter, wherein the fluid is passed through the first filter and the second filter.

19. A kit for detecting at least one component in a fluid in situ, comprising:

(a) a first filter and a second filter; and

(b) a biosensor, wherein the fluid is passed through the first filter and the second filter, thereby forming a concentrated test sample, and wherein the concentrated test sample is contacted with the biosensor, said biosensor detecting the at least one component.

20. A device for concentrating at least one component in a fluid, comprising: a first reservoir; a second reservoir; a first filter; a third reservoir; and a second filter, wherein the fluid is drawn from the first reservoir into the second reservoir and passed through the first filter, thereby forming a first concentration sample in the second reservoir; and wherein the first concentration sample is drawn from the second reservoir into the third reservoir and passed through the second filter, thereby forming a second concentration sample in the third reservoir.

21. The device according to claim 20 wherein the first reservoir is approximately 5OL in capacity, the second reservoir is approximately 2L in capacity, and the third reservoir is approximately 20OmL in capacity.

22. The device according to claim 20 or claim 21 , wherein the at least one component is selected from the group comprising eukaryotic or prokaryotic organisms, cells, organelles, plants, yeasts, algae, protozoa, bacteria, mycoplasma, viruses, prions, proteins, peptides, polypeptides, immunoglobulins, biotin, substrates, enzymes, receptors, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, lipids, nucleic acids, macromolecules or any other molecule or any combination, fraction or part thereof.

23. The device according to claim 22, wherein the fluid may comprise any mixture, suspension, dispersion, solution or combination thereof.

24. The device according to claim 23, wherein the fluid is selected from the group comprising water, including river water, drinking water, catchment water, seawater, artesian water and bore water, sewerage, effluent, fermentation broths, liquids, cell lysates, cell culture supernatants, cell extracts, cell suspensions, protozoan cultures or lysates, bacterial cultures or lysates, viral cultures or lysates, plant extracts or any combination, fraction or part thereof.

25. The device according to claim 20 or claim 21, further comprising a data collection device.

26. The device according to claim 25, wherein the data collection device may collect data in real time or as logged data.

27. The device according to claim 25, further comprising a power supply.

28. The device according to claim 20 or claim 21, further comprising a self-cleaning module.

29. The device according to claim 27 or claim 28, whereby the forming of the first and second concentration samples is automated.

30. A method for concentrating at least one component in a fluid, comprising: drawing the fluid from a first reservoir into a second reservoir and passing it through a first filter, thereby forming a first concentration sample in the second reservoir; and drawing the first concentration sample from the second reservoir into a third reservoir and passing it through a second filter, thereby forming a second concentration sample in the third reservoir.

5 31. A device for detecting at least one component in a fluid comprising: a first reservoir; a second reservoir; a first filter; a third reservoir; o a second filter; and a biosensor, wherein the fluid is drawn from the first reservoir into the second reservoir and passed - through the first filter, thereby forming a first concentrated sample in the second reservoir; wherein the first concentrated sample is drawn from the second reservoir into the thirds reservoir and passed through the second filter, thereby forming a second concentrated sample in the third reservoir; and wherein the second concentrated sample is contacted with the biosensor, said biosensor detecting the at least one component. o

32. The device according to claim 31 wherein the first reservoir is approximately 5OL in capacity, the second reservoir is approximately 2L in capacity, and the third reservoir is approximately 20OmL in capacity.

33. The device according to claim 31 or claim 32, wherein the at least one component₅ is selected from the group comprising eukaryotic or prokaryotic organisms, cells, organelles, plants, yeasts, algae, protozoa, bacteria, mycoplasma, viruses, prions, proteins, peptides, polypeptides, immunoglobulins, biotin, substrates, enzymes, receptors, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, lipids, nucleic acids, macromolecules or any other molecule or any combination, fraction or part thereof. 0

34. The device according to claim 33, wherein the fluid may comprise any mixture, suspension, dispersion, solution or combination thereof.

35. The device according to claim 34, wherein the fluid is selected from the group₅ comprising water, including river water, drinking water, catchment water, seawater, artesian water and bore water, sewerage, effluent, fermentation broths, liquids, cell lysates, cell culture supematants, cell extracts, cell suspensions, protozoan cultures or lysates, bacterial cultures or lysates, viral cultures or lysates, plant extracts or any combination, fraction or part thereof.

36. The device according to claim 31 or claim 32, further comprising a data collection device.

37. The device according to claim 36, wherein the data collection device may collect data in real time or as logged data.

38. The device according to claim 36, further comprising a power supply.

39. The device according to claim 31 or claim 32, further comprising a self-cleaning module.

40. The device according to claim 38 or claim 39, whereby the forming of the first and second concentration samples is automated.

41. A method for detecting at least one component in a fluid, comprising: drawing the fluid from a first reservoir into a second reservoir and passing it through a first filter, thereby forming a first concentration sample in the second reservoir; drawing the first concentration sample from the second reservoir into a third reservoir and passing it through a second filter, thereby forming a second concentration sample in the third reservoir; and contacting the second concentration sample with a biosensor, thereby detecting the at least one component in the fluid.