Abstract:
An in-line water monitoring system for the detection of the accidental or intentional introduction of potentially harmful substances. The automated system comprises a water pressure driven concentration unit that filters drinking water through a hollow-fiber filter. Material collected on the filter is backflushed into a collection vessel by passing a sterile solution through the filter in the reverse direction. An electronic signal at the end of the backflush sequence triggers a sensor such as an array biosensor to begin processing and analyzing the sample. The array biosensor houses a slide prepared with antibodies to the test organism. The array biosensor is programmed to automatically run sample and detection reagents over the slide, analyze the resulting pattern for positive and negative data, and report the results.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of International patent Application PCT/US2006/006002 filed Feb. 18, 2006 which claims priority to U.S. Provisional Patent Application 60/593,484, filed Feb. 18, 2005; which is fully incorporated herein by reference. 
     
    
     GOVERNMENT SUPPORT 
       [0002]    This invention was developed under support from: the U.S. Army Research, Development and Engineering Command (RDECOM) under grant DAAD13-00-C-0037, accordingly the U.S. government may have certain rights in the invention; and Pinellas County Utilities under grant 1209-101-700, who may have certain rights in the invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    The safety of drinking water has long been a concern of water utilities and other government entities. Current analysis methods take several days to accomplish and there is a desire for more rapid methods of determining when a potential health hazard is present in a water supply. In addition, potable water supplies are considered part of the U.S critical infrastructure that has been mandated to increase security since Sep. 11, 2001. Military services are also concerned about the security of this critical resource at military bases and temporary field military installations. 
         [0004]    The prior art describes methods using hollow-fiber filter ultra-filtration to concentrate microorganisms from water for subsequent detection. Previous methods, however, require manual control of the system; none are amenable to being automated. Previous attempts to detect the presence of microorganisms require the sample to be transported to a remote location to be tested. Existing systems also require pretreatment of the filter prior to concentration in order to achieve adequate concentration of the targeted microorganisms. Pre-treatment increases the complexity of the concentration process and prevents automation of the system. 
         [0005]    Therefore, what is needed is an automated device that is capable of being placed online in a flow system to monitor for the presence of microorganisms. 
       SUMMARY OF INVENTION 
       [0006]    This invention provides a method of concentrating hazardous biological material, including bacteria, viruses and toxins, from water sources. The concentrator may be coupled to a sensor that screens the concentrate for the presence of designated hazardous substances. Users can continuously concentrate potentially hazardous materials from a water source for a desired amount of time by placing it in the water flow path or by diverting a subset of the water flow to the concentrator. For example, the device could be placed in the public drinking water distribution system and used to monitor the security of this critical resource. While the protection of potable water resources provides the broadest benefit, other types of water or liquid streams can also be monitored using this technology and multiple uses are contemplated. 
         [0007]    The inventive system includes an on-line water concentration system to facilitate the detection of potentially harmful substances. The automated system comprises a water pressure driven concentration unit that filters drinking water through a hollow-fiber filter. Material collected on the filter is backflushed into a collection vessel by passing a sterile solution through the filter in the reverse direction. An electronic signal can be delivered at the end of the backflush sequence to trigger a sensor, such as an array biosensor, to begin processing and analyzing the sample. The array biosensor houses a slide prepared with antibodies to the test organism. The array biosensor is programmed to automatically run sample and detection reagents over the slide, analyze the resulting pattern for positive and negative data, and report the results. 
         [0008]    The inventive system removes any hazardous material suspended in the fluid that is greater than the pore size of the filter to create a concentrate. The use of subsystems makes filter pretreatment unnecessary. Analysis of the concentrate thereby alerts a user to any hazardous material discovered and identified. The process is automated and requires an attendant where a harmful material is discovered or if maintenance is required. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]    For a fuller understanding of the nature and objects of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: 
           [0010]      FIG. 1  is a schematic representation of the invention showing the integrated system. 
           [0011]      FIG. 2  is a schematic representation of the invention showing the flow path of the forward flow concentration subsystem. 
           [0012]      FIG. 3A  is a schematic representation of the invention showing the flow path of the air flush subsystem. 
           [0013]      FIG. 3B  is a schematic representation of the invention showing the flow path of the liquid backflush subsystem. 
           [0014]      FIG. 4  is a schematic representation of the invention showing the flow path of the cleaning subsystem. 
           [0015]      FIG. 5  is a schematic representation of the invention showing the flow path of the purge subsystem. 
           [0016]      FIG. 6A  is a table of data from experiments using the inventive method. 
           [0017]      FIG. 6B  is a table of data from experiments using the inventive method. 
           [0018]      FIG. 6C  is a table of data from experiments using the inventive method. 
           [0019]      FIG. 7  is a table of data from experiments using the inventive method. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0020]    In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part hereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. 
         [0021]    The concentration system filters particulate matter that is larger than the pore size of the filter from a liquid stream. Particulate matter collects within the hollow cores of the filter fibers. The collected particulate material is recovered by back-flushing the filter with a predetermined volume of liquid such as water, buffer or other solution. The concentration of collected particulate matter (e.g., bacteria, viruses, toxins) is much greater in the recovered concentrate than in the original water source. The concentrate may be directed to a sensor for detection and identification of its constituents. The inventive system also includes a cleaning function that washes the filter after every concentration cycle and readies the filter to start a new cycle. The entire process is automated and controlled by a programmable logic controller. The programmable logic controller can be equipped with software tailored to the system&#39;s intended use. Examples of programmable variables include, inter alia, collection time, purge delay and time, volume of backflush solution, cleaning time and delivery of the concentrate sample to a biosensor for detection. 
         [0022]    One embodiment of the inventive system employs a filter capable of processing large volumes of water. By way of example only, one embodiment uses a unique filter produced by Norit Membrane Technology Bv (Netherlands) that is amenable to processing large volumes of water. The ideal filter has backflush capabilities. Backflushing of the filter removes particulate matter collected on the interior of the filter fibers. Backflushing also accommodates periodic cleaning of the filter, thereby extending filter-life. The process of filtering and removal of particulates from an ultrafilter via backflushing is referred to as dead end ultrafiltration. 
         [0023]    The following represents an illustrative device developed based on the methods of the inventive system. This example represents only one filtration device that permits concentration of particles, including microorganisms, from the liquid flow according to the inventive method. 
         [0024]    Referring now to the figures,  FIG. 1  shows a schematic view of an illustrative device. Discussion of this particular embodiment will lend a greater understanding of the inventive method, although other embodiments are contemplated. Automated Concentration System (ACS)  1  is best understood when viewed in light of its modular elements. ACS  1  comprises forward-flow concentration subsystem  10 , backflush subsystem  50 , cleaning subsystem  100  and purge subsystem  120 . Backflush subsystem  50  further comprises liquid-backflush subsystem  50   a  ( FIG. 3B ) and air-backflush subsystem  50   b  ( FIG. 3A ). 
         [0025]    Programmable Logic Controller (PLC) 
         [0026]    Automation of the inventive system is possible with the use of a programmable logic controller (PLC). The term programmable logic controller (or PLC) as used herein is any device used for the automation of the disclosed system. While the PLC usually will incorporate a microprocessor, device relying on mechanical control (i.e. timers) are also contemplated. In a preferred embodiment the PLC remains in electronic communication with the consituent elements of the system, including sensors, valves, solenoids, pumps, gauges and actuators. The input/output arrangements necessary to practice the invention may be built into a simple PLC, or the PLC may have external input/output modules attached to a proprietary computer network that plugs into the PLC. Although the current system is optimized for automation, manual operation is also envisioned. 
         [0027]    In a preferred embodiment the PLC is equipped with software that provides an interface for control of forward flow (concentration) time, purge delay and length, interior filter drain time, number of air flushes, number of backflush sequences, cleaning solution circulation time and cleaning solution flush sequence and time. A system diagram incorporated into the user interface can provide feedback on flow paths during operation. Controls may also be provided to configure the system for introduction of a sample to test the operation of the system. An assay recipe program directs the sequence of concentration steps. The recipe program includes a choice of standard concentration processes or provides flexibility by allowing the user to encode a different sequence, if desired, prior to initiating the concentration process. 
         [0028]    The PLC controls flow through the system by opening and closing solenoid valves, S 1  through S 5 , located at strategic points on the system. In the cleaning sequence shown in  FIG. 5 , for example, the PLC would open solenoid valves S 3  and S 4  but close solenoid valves S 1 , S 2  and S 5  (see  FIG. 1 ). A check valve can be incorporated to prevent the introduction of fluid into the backflush subsystem. 
         [0029]    Forward-Flow Concentration Subsystem (FFC) 
         [0030]    Forward-flow concentration subsystem (FFC)  10 , shown in  FIG. 2 , includes filter housing  35 , containing hollow fiber filter  30 , with a support structure that permits water to be pushed through the filter using only the pressure from source line  15 . The direction of water flow through FFC  10  is indicated by arrow A 1 . In one embodiment flow to filter  30  is controlled by ball valve  20 , to turn flow on and off, and needle valve  25  to adjust the pressure of the water into filter  30 . An optional pre-filter, not shown, may be installed to remove large particulates that could clog filter  30  in applications involving relatively dirty water. For example, a pre-filter may be installed between ball valve  20  and needle valve  25 . 
         [0031]    Water is directed into the interior of the hollow fibers of filter  30  wherein particles larger than the pore size of the filter are retained within the fiber cores and all other material passes to the exterior space of filter cartridge  35 . Accordingly, the pore size of the filter can be selected to target a specific type of pathogen or particulate matter. In the embodiment shown in  FIG. 2 , water continues to flow to drain  40 . In alternate embodiments, water and material not trapped by filter  30  is discarded or is transferred back to the source flow line or an alternate location. Optional spiking port  45  allows a user to introduce a sample; e.g. to test system operation. 
         [0032]    Backflush Subsystem 
         [0033]    The programmable logic controller (PLC) initiates backflush subsystem  50  after a predetermined amount of water passes through filter  30 . The PLC turns off water flow to filter  30  prior to engaging a backflush sequence. Backflush subsystem  50  permits either a gravity drain of the fiber cores, an air-flush of the fiber cores ( FIG. 3A ) or a liquid backflush of a solution of choice ( FIG. 3B ) through the fiber to remove particulate material trapped within filter  30 . In this embodiment, both air-backflush subsystem  50   a  and liquid-backflush subsystem  50   b  use a 50-ml syringe pump. While other mechanisms can be used, the use of a syringe pump for backflush sequences provides better control over the backflush sequence and concentrate collection process. The gravity drain function is accomplished by opening solenoid valves located on the top and bottom of the filter blocks that hold filter cartridge  35  in position. The sequence of the three backflush options are programmed into, and controlled by, the PLC. Particulate matter released from filter  30  passes through sample-drain  41  and is collected in collection vessel  65 . Material in collection vessel  65  is delivered to biosensor  70  for detection and identification of particulates. A pressure gauge is located in a position that permits measurement of the backflush pressure. 
         [0034]    Air-backflush subsystem  50   a  is outlined in  FIG. 3A . Although this embodiment uses ambient air to flush the system, any fluid can be incorporated and the selection of an appropriate gas will require an analysis of the intended use of the system. Here, the PLC initiates an air-backflush sequence thereby starting pump  55 , which then draws air through air-valve  75 . The air then travels under pressure along path of travel A 2  through filter  30 , thereby removing liquid from the fiber cores along with some particulate matter trapped therein. The sample continues along path of travel A 2  through sample-drain  41  into collection vessel  65 . The sample can be directed from vessel  65  to the optional biosensor  70  responsive to a signal from the PLC. Parameters governing delivery to the biosensor are varied but can include time and or volume. Useful biosensors are known and will be apparent to one skilled in the art considering factors such as the particulate matter being analyzed and the intended use of the system. Examples of useful biosensors include the RAPTOR (Research International, Inc.) and the ACA-ABS (Constellation Technology Corporation). 
         [0035]    Liquid flow through liquid-backflush subsystem  50   b , detailed in  FIG. 3B , is shown by directional arrows B 1  and A 3 . Solution reservoir  60  is placed in fluid communication with syringe pump  55 . Solution reservoir  60  can be filled with any liquid, the selection of which may vary depending on the system&#39;s intended use. Commonly, reservoir  60  will be filled with a predetermined quantity of water, buffer or other solution. Reservoir  60  can also be placed in fluid communication with a source of the selected liquid thereby enhancing the system&#39;s automation. The liquid-backflush sequence is initiated by the PLC which starts pump  55 . Solution is drawn from reservoir  60  along path of travel B 1  to pump  55 . From pump  55  the solution continues along path of travel A 3  through filter  30  from the cartridge space to the inside of the fiber cores, thereby removing any concentrated particulate matter trapped therein to form a sample. The sample continues along path of travel A 3  through sample-drain  41  into collection vessel  65 . The sample can be directed from vesicle  65  to biosensor  70  responsive to a signal from the PLC. Parameters governing delivery to the biosensor are varied but can include time and or volume. 
         [0036]    The inventive method is not limited by any one sequence of events. The clearing of the fiber cores in filter  30  with air before backflushing the filter with liquid, however, enhances the efficiency of the backflush step. 
         [0037]    Cleaning Subsystem 
         [0038]    The cleaning sequence initiates responsive to a signal from the PLC once the particulate matter in filter  30  has been backflushed into the collection vesicle. Cleaning solution reservoir  105  incorporates a precision temperature control device. In this illustrative embodiment reservoir  105  holds up to 5 liters of cleaning solution at a user-determined temperature. Cleaning subsystem  100  sequence circulates the heated cleaning solution through filter  30  in the forward flow path of travel (A 4 ). A cleaning cycle is completed when the cleaning solution returns to reservoir  105 , but multiple cleaning cycles can be incorporated into a single cleaning sequence. The type of solution, cleaning temperature and length of cleaning cycle are determined by the user. The cleaning solution is removed from filter  30  and system lines by a combination of forward flow and backflush events initiated by the PLC. 
         [0039]    A new forward flow concentration cycle is started upon the successful completion of the cleaning sequence. If desired, two or more units can be linked to the source flow and collection alternated between the two units. Redundant use of the inventive system ensures that one unit is operational while the other is being cleaned thereby eliminating gaps in collection. 
         [0040]    Purge Subsystem 
         [0041]    Purge subsystem  120 ,  FIG. 5 , comprises purge valve  125  and purge reservoir  130 . Purge valve  125  and purge reservoir  130  are optimally positioned at the top of filter cartridge  35  to permit the escape of any air or gas that has collected within filter cartridge  35 . This safety features prevents flow shutdown due to air pressure buildup at the outflow point of filter cartridge  35 . Pressure gauges located on the inlets and outlets of filter cartridge  35  permit the pressure across the membrane to be monitored. 
       Example I 
       [0042]    The following makes reference to the test data provided in  FIGS. 6A through 6C . 
         [0043]    Runs 1 &amp; 2 ( FIG. 6A ). 
         [0044]    A new 0.8 mm Norit filter or a used filter that had been soaking in 1% bisulfite solution preservative was used for the each test. The filter was installed and washed with water from the faucet, which was fed by drinking water. The filter was then backflushed with distilled water. The pH of permeate and recovered backflush liquid was measured during cleaning to ensure that the bisulfite was removed from the filter prior to beginning a concentration run. Prior to spiking with microspheres, water was run through the filter in the forward direction for 5-7 minutes and the transmembrane pressure and flow rate were measured. 
         [0045]    For the tests, 700 μl of a 2.733×10 8  spheres/ml (in phosphate buffer, pH 7.4) concentration of fluorescent microspheres (1 μm, carboxylate-modified, yellow-green FluoSpheres, Molecular Probes, Eugene, Oreg.) were diluted into 10 mls distilled water and injected into the concentrator using the sample injection port and with the system in “spike” mode. The microspheres were followed by 10 additional mls of water to wash them completely into the system. Forward flow was initiated and timed for 5 minutes of flow. The transmembrane pressure and flow rate were monitored during the concentration. Total recovery was better in the liquid/liquid (Run2) backflush experiment, but the concentration of the recovered material was higher in the liquid/air experiment in fractions collected after the air push. 
         [0046]    The filter was back flushed using the following procedure: 
         [0047]    Run 1—purge drain (to dump purge volume back into column), syringe air push through fiber centers ×1, syringe phosphate buffer backflush ×4 (water/air); and 
         [0048]    Run 2—purge drain, syringe air push backflush (outside to inside of fibers)×1, syringe phosphate buffer backflush ×2 (water/water). 
         [0049]    Runs 3 &amp; 4 ( FIG. 6B ) 
         [0050]    The procedure was similar to the previous tests, discussed above, except 400 μl of microspheres were spiked into the concentrator and permeate was used to dilute them instead of distilled water. The previously used filter that had been stored in bisulfite was used for the first test. The second test used a new filter. Both filters were rinsed with forward flow and backflush to rinse out bisulfite (and glycerin in the new filter). For both runs, the following fractions were collected: purge drain, syringe air push through fiber centers ×1, phosphate buffer backflush ×3. 
         [0051]    These tests support results from the previous test showing good concentration (10 6  spheres/ml) when the fiber centers were cleared with air prior to backflushing with phosphate buffer. The greater than 100% recovery calculated for the runs may be attributed to either microsphere accumulation on the filter or miscalculation of the spike concentration. 
         [0052]    Run 5 ( FIG. 6C ) 
         [0053]    The filter from the last microsphere run (new filter), which had been stored in bisulfite, was used. This filter was used for one microsphere run but had never been used with spores and never been cleaned using the hot NaOH procedure. A stock suspension of  B. globigii  spores with an average of 1.25×10 8  spores/ml (n=2) was prepared. The stock suspension was more difficult to count this time because of the presence of unidentified junk in the suspension. One milliliter of this suspension was used to spike the filter using the same procedure described above for the microspheres. Concentrations of collected fractions were determined using both direct counts and enumeration plating. Plates done the day of the experiment were difficult to interpret so additional plates, all at the same dilution of 10 −2 , were prepared in an attempt to get a better feel for the relative concentration of each fraction. 
         [0054]    The counts for this concentration presented difficulties because there was little consistency among the three attempts at enumeration. Direct counts were difficult to obtain due to the presence of a large amount of particulates, making the counts unreliable. Note that the direct count of spores is less than counts based on plates and that the total recovery for the fractions is greater than 100%. These indicated that the direct count may not be accurate. The stock used for this experiment was stored in a desiccator cabinet at room temperature and those used previously were stored in a refrigerator. 
       Example II 
       [0055]      FIG. 7  shows the results of several tests of the inventive system after it was fully automated and connected to a WAMO ABS and/or WAMO TDU. All runs were done using the same protocol for recovering sample (concentrate) from the filter. The backflush solution was sterile deionized water. Spore concentrations were based on viable counts on TSA and are expressed as CFU/mL. Although this method is known to underestimate spore concentration because not all the spores will germinate, it was better than direct microscopic counts because particulates in the concentrate made it impossible to accurately identify and count spores. Improved methods of calculating spore concentrations are being investigated. Experiment #11 is a continuous concentration experiment in which the concentrator was programmed to run in a repetitive mode, consisting of 6-hour concentration intervals followed by sample recovery, for approximately 3½ days. Near the end of a 6 hour concentration period, the system was spiked with  B. globigii  upstream from a water softener prefilter and forward flow resumed for an additional 2 hours. All other tests were done using 15 minute forward flow times after spiking using port  45 . 
         [0056]    Referring to  FIG. 7 , concentrate was collected in fractions in experiments 2 through 4. The concentration of each fraction was multiplied by the volume of the fraction to get total CFU in the fraction; the total CFUs for each were summed and divided by the total volume of all fractions to calculate a concentration for the collected material. Water volume was calculated by averaging flow rates over the time of the concentration run and multiplying by the total run time. Water volume for experiment 11 only includes the water that flowed through the filter after the system was spiked with  B. globigii . The ACS performed relatively consistently considering that  B. globigii  is known to give somewhat inconsistent recovery from filters. Recoveries calculated ranged from about 1-68%, with most (5/11) in the 20-30% range. Concentration factors ranged from 3-56 fold. Concentrations in the recovered material ranged from approximately 3×10 4  to 1×10 6  CFU/mL and were all detectable on the biosensor, although the positives from Experiment  9  were only faintly fluorescent. The variability could also be due to the inconsistency of viable counts. Not all  B. globigii  spores will germinate and the percent that do can vary greatly. Normally, viable counts are 0.5 to 1 log less than direct counts. 
         [0057]    It will be seen that the objects set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
         [0058]    It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall there between. Now that the invention has been described,