Abstract:
A biological sample collector is adapted to a collect several biological samples in a plurality of filter wells. A biological sample collector may comprise a manifold plate for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the tube to deposit a biological sample onto a filter in the bottom of the selected filter well. The biological sample collector may be portable.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the priority of U.S. Provisional Application No. 60/773,732, filed on Feb. 14, 2006, the entire disclosure and contents of which is hereby incorporated by reference. 
    
    
     GOVERNMENT INTEREST STATEMENT 
     The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of the Lawrence Livermore National Laboratory. 
    
    
     BACKGROUND 
     1. Field of the Invention 
     The present invention relates generally to biological sample collectors, and more particularly to biological sample collectors used in verifying a biological decontamination protocol. 
     2. Related Art 
     After an attack involving a biological agent against a facility, authorities must act to decontaminate if the facility will be used by the public again. A variety of biological decontamination protocols may be used to clean a contaminated facility. Even though these protocols exist, the assessment of the effectiveness of a selected protocol may be time-consuming and difficult due in part to the range of weaponizable biological agents. Moreover, the correct tools and methods for identifying the presence of many biological agents are unknown, which also may slow the verification of the decontamination protocol. Furthermore, biological agents may not have a uniform susceptibility to a particular decontamination protocol. 
     One important aspect in verifying the efficiency of the decontamination protocol is the collection of biological samples from the environment. Samples must be collected from different surfaces within the environment and tested. Conventional devices and tools for collecting are insufficient and not suitable for all types of surfaces. For example, wipes are not suitable for collecting samples from carpet or fabric and are easily tearable. Further, the biological samples collected using these conventional devices and tools are not suited to be directly tested using a high-throughput automated process. 
     SUMMARY 
     According to a first broad aspect of the present invention, there is provided a biological sample collector comprising a manifold plate for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the tube to deposit a biological sample onto a filter in the bottom of the selected filter well. 
     According to a second broad aspect of the invention, there is provided a biological sample collector comprising a suction means in fluid communication with a filter plate, wherein the filter plate comprises a plurality of filter wells; a hollow slider for engaging and positioning a tube that slides therethrough; and a slide case within which the hollow slider travels to allow the tube to be aligned with a selected filter well of the plurality of filter wells, wherein when the tube is aligned with the selected filter well, the tube is pushed through the hollow slider and into the selected filter well to sealingly engage the selected filter well and to allow the suction means to pull a biological sample from the tube that is deposited on a filter in the bottom of the selected filter well. 
     According to a third broad aspect of the invention, there is provided a biological sample collector comprising a suction means within a housing; a manifold plate mounted on the housing for mounting a filter plate thereon, the filter plate having a plurality of filter wells therein; and a tube case having a plurality of tube holders each for engaging and positioning a tube that slides through the selected tube holder, wherein when the tube is aligned with a selected filter well of the plurality of filter wells, the tube is pushed through the selected tube holder and into the selected filter well to sealingly engage the selected filter well and to allow the suction means to pull from the tube a biological sample that is deposited on a filter in the bottom of the selected filter well. 
     According to a fourth broad aspect of the invention, there is provided a method for collecting one or more biological samples comprising the following steps: (a) inserting a tube into a first filter well; (b) transferring through the tube a first set of biological samples onto a first filter in said first filter well; (c) moving the tube into alignment with a second filter well; and (d) transferring through the tube a second set of biological samples onto a second filter in said second filter well. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will be described in conjunction with the accompanying drawings, in which: 
         FIG. 1  is an exploded side view of a biological sample collector in accordance with an embodiment of the present invention; 
         FIG. 2  is a perspective view of a biological sample collector having a slide case in accordance with an embodiment of the present invention; 
         FIG. 3  is a perspective view of a biological sample collector having a tube case in accordance with an embodiment of the present invention; 
         FIG. 4  is a side view of an assembled biological collector having a gun-shaped housing in accordance with an embodiment of the present invention. 
         FIG. 5A  is a perspective view of a slide case in accordance with an embodiment of the present invention; 
         FIG. 5B  is a cross-section view of a hollow slider within a slide case in accordance with an embodiment of the present invention; 
         FIG. 5C  is a perspective view of a hollow slider in accordance with an embodiment of the present invention; 
         FIG. 5D  is a perspective view of a hollow slider with the clamps retracted in accordance with an embodiment of the present invention; 
         FIG. 5E  is a perspective view of a hollow slider with the clamps pushed down in accordance with an embodiment of the present invention; 
         FIG. 6  is a detailed cross-section view of a tube aligned with a filter well in accordance with the embodiment shown in  FIG. 1 ; 
         FIG. 7  is a top view of a film barrier in accordance with an embodiment of the present invention; 
         FIG. 8A  is a side view of an assembled biological collector having a tube aligned with one filter well in accordance the embodiment shown in  FIG. 1 ; and 
         FIG. 8B  is a side view of an assembled biological collector of  FIG. 8A  where the tube is aligned with a second filter well in accordance with the embodiment shown in  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION 
     It is advantageous to define several terms before describing the invention. It should be appreciated that the following definitions are used throughout this application. Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated. 
     DEFINITIONS 
     For the purposes of the present invention, the term “biological agent” refers to a biological organism that is present in an environment that is not normally present, or whose presence is at levels that are higher than normal, and whose reduction, elimination, destruction or neutralization is desirable. Biological agents include existing and yet to be developed biological weapons. The range of biological organisms includes spores, bacteria, prokaryotes, viruses, yeasts, fungi, and other single cell organisms. One example of a biological agent is an anthrax,  bacillus anthracis , spore. Note that when this application refers to collecting biological agents, this application is referring to collecting multiple biological organisms of one type of biological agent, unless otherwise indicated. 
     For the purposes of the present invention, the term “biological sample” refers to the material, organisms and debris collected from an environment. The biological sample may have one or more biological agents present. 
     For the purposes of the present invention, the term “decontamination protocol” refers to any method of cleaning, destroying, removing or killing a biological contamination from an environment. The decontamination protocol may involve a vapor, solution or physical decontamination, or combinations thereof. Examples of biological decontamination protocols include fumigation with chlorine dioxide, steam sterilization, or gamma radiation. Some current methods of decontamination involve using combinations of solutions or fumigants of sodium hypochlorite (bleach), ethylene oxide, methyl bromide, ozone, paraformaldehyde, and other antiseptics and disinfectants, as well as vacuum cleaning with HEPA filtration. Further description of decontamination protocols are provided in Berns, et al., “Reopening Public Facilities After a Biological Attack: A Decision-Making Framework,” National Academies Press (2005), the entire contents and disclosures of which is hereby incorporated by reference. 
     For the purposes of the present invention, the term “environment” refers to a facility, building, or transport device where the presence of a biological agent has been detected. A treated environment refers to a facility or building that is in the process of being or has been cleaned using biological decontamination protocols. It should be readily understood that environment may encompass a plurality of separate buildings in an area. A transport device includes trains, buses, trolleys, subways, airplanes, ferries, cruise ships, barges, cargo ships or similar vehicles used in transportation of people and goods. 
     For the purposes of the present invention, the term “fluid communication” or “fluidly connected” refers to an interaction between two or more components in which a fluid is used. The fluid may comprise a liquid, gas or mixture thereof. For example the air pump is fluidly connected, using air, to the tube through the network of pipes in the manifold plate and filter well. 
     For the purposes of the present invention, the term “polymerase chain reaction” (PCR) refers to a method of amplifying a specific target polynucleotide through repeated cycles of DNA strand separation (denaturating), annealing of oligonucleotide primers, and DNA polymerization using a thermostable DNA polymerase. As used herein, “quantitative polymerase chain reaction” (qPCR) refers to a PCR reaction performed such that the assay is capable of quantifying the amount of target polynucleotide present in the sample. Also, as used herein “rapid viability-PCR” (RV-PCR) refers to a qPCR that determines whether a sample is viable within approximately 24 hours or less. 
     For the purposes of the present invention, the term “sealingly” refers to the interaction when two components are connected or engaged as being substantially air tight. Although components may be sealingly engaged when assembled, the components may be separated and reattached. 
     For the purposes of the present invention, the term “viable” refers to the ability of a biological organism, including biological agents, to carry out those biochemical and genetic processes that allow the organism to propagate under suitable conditions, including gene expression, i.e. transcription, and DNA and RNA replication. Organisms that require the presence of a host cell in order to propagate are considered to be “viable” so long as they are capable of propagation in the presence of a suitable host cell. Moreover, for organisms, such as viruses, and certain mycoplasmata, viability necessarily connotes infectivity, i.e., without the ability to infect a compatible host cell, such agents would be considered non-viable. 
     DESCRIPTION 
     To rapidly restore a facility following a biological agent release, i.e. in a terrorist attack, the verification method of the decontamination protocol must provide quick turn around time and reliable results to determine building safety. Aspects of the present invention provide devices and methods to collect biological samples for verifying the biological decontamination protocols using highly specific, quantitative, real-time, polymerase chain reaction (PCR) assays. The effectiveness of a protocol may be determined by whether any collected biological agents are viable after treatment using the protocol. The viability of the biological agents collected from an environment corresponds to the viability of the possible biological agents remaining in the environment. Once an effective biological decontamination protocol is verified, the authorities may confirm that a facility is clean enough to be safe. 
     To collect the biological agents, embodiments of the present invention provide a biological sample collector, also referred to as an Enviro CAF™. The biological sample collector may be used in both indoor and outdoor environments. One important aspect of the biological sample collector is that the collection is optimized such that the biological samples are collected directly in a filter well. This allows culturing and further testing to occur within the filter well without the need to transfer the biological sample. One advantage of such embodiments is that testing occurs within the device used to collect the biological sample. An example of testing method is described in co-pending applications entitled, “Rapid Viability Assessment of Biological Indicators by qPCR,” filed on Feb. 13, 2007, Ser. No. 11/706,064, now abandoned and “Rapid Viability Assessment of Environment Samples by qPCR,” filed on Feb. 13, 2007, Ser. No. 11/706,063, the entire contents and disclosures of which are hereby incorporated by reference. Such embodiments increase safety and reduce unnecessary contamination or aerosolization when collecting a biological agent. 
     Referring now to the embodiment depicted in  FIG. 1 , the biological sample collector  100  comprises a housing  102 , manifold plate  104 , filter plate  106  and slide case  108 . Housing  102  comprises an actuator  112 , air pump  114 , notch  116 , base extension  118 , and a power port  120 . Air pump  114  has a conduit  122  that opens out of housing  102  and air pump  114  is adjacent to vent grate  124 . On base extension  118  there is a clamp  126 . Connected to housing  102  through power port  120  is a power source  128  having an activation switch  130 . Manifold plate  104  comprises a casing  132  that surrounds a pipe  134  having an outlet valve  136  and a plurality of intake valves  138 . Filter plate  106  comprises a plurality of filter wells  140  in a strip  142 . Each filter well  140  comprises a trap  144 , shaft  146 , filter  148  and tapered end  150 . Positioned above trap  144  is a film barrier  152 , described in further detail below with reference to  FIG. 7 . A tube  160  is inserted through the slide case  108 . Slide case  108  comprises a lid  162  having grooves  168  for guiding hollow slider  170 , described in further detail below with reference to  FIGS. 5A-5E . Hollow slider  170  has an empty bore cylinder  176  through which tube  160  travels. Lid  162  also has a tenon  186  that engages notch  116  when biological sample collector  102  is assembled. 
     The embodiment depicted in  FIG. 2  show a biological sample collector  200  comprising a housing  202  having a manifold plate  204  and filter plate  206  mounted thereon, as well slide case  208 . Housing  202  comprises an actuator (not shown) on handle  213 , air pump  214 , base extension  218 , and a power port  220 . Air pump  214  has a conduit  222  that opens out of housing  202 . On base extension  218  there is a clamp  226 . Connected to housing  202  through power port  220  is a power source (not shown). Manifold plate  204  comprises a casing  232  that surrounds a pipe  234  having an outlet valve  236  and a plurality of intake valves  238 . Filter plate  206  comprises a plurality of filter wells  240  in a strip  242 . Each filter well  240  comprises a shaft  246 , filter  248  and tapered end  250 . A tube  260  is inserted through slide case  208 . Slide case  208  comprises a lid  262  having an enclosed space  264  in which hollow slider  270  fits. Hollow slider  270  has an empty bore cylinder  276  through which tube  260  travels. Hollow slider  270  is shown in position A′ removed from slide case  208  and position A″ at an incline for loading into, slide case  208 . Hollow slider  270  has teeth  284  for engaging corresponding teeth  285  on filter plate  206 . Once engaged the teeth  284  and  285  may align and position hollow slider  270  to allow tube  260  to be inserted into filter well  240 . Air may flow from tube  260  to air pump  214  as shown by arrows  286 . 
     The embodiment in  FIG. 3  shows a biological sample collector  300  comprising a housing  302 , combined filter plate  305  and tube case  310 . Housing  302  comprises an actuator (not shown), air pump  314 , notch  316 , base extension  318 , and a power port  320 . Air pump  314  has a conduit  322  that opens out of housing  302 . On base extension  318  there is a clamp  326 . Connected to housing  302  through power port  320  is a power source  328  having a clip  329 . Combined filter plate  305  comprises a manifold pipe  334  having an outlet valve  336  and a plurality of intake valves  338  and a plurality of filter wells  340  aligned with intake valves  338 . Each filter well  340  comprises a shaft  346 , filter  348  and tapered end  350 . Positioned above filter well  340  is a film barrier  352 , described in further detail below with reference to  FIG. 7 . Tube case  310  comprises a lid  354  and tube holders  356  that extend through lid  354 . Lid  354  has a tenon  358  that is inserted into notch  316  when biological sample collector  302  is assembled. This closed mortise-tenon joint aligns and secures lid  354  to housing  302 . The number of tube holders  356  corresponds to the number of filter wells  340  in filter plate  306 . Tube  360  is removably inserted in one of the tube holders  356 . Air may flow from tube  360  to air pump  314  as shown by arrows  386 . 
     In the embodiments of the present invention, a manifold plate is mounted to housing, filter plate is mounted to manifold plate, and slide case or tube case is mounted on filter plate and housing. These plate and case components are modular and may be interchanged or removed. For example filter plate may be removed once all the biological agents are deposited and a new filter plate may be inserted. Several different types of filter plates may be used, including 6, 8, 12, 48 or 96 filter-well plates. It should be understood that various combinations of the 8 and 12 filters well strips may be assembled into a 48 or 96 filter-well plate for use with robotic testing systems and devices. Depending on the filter plate, the size and configuration of the manifold plate may vary. In some embodiments of the present invention, such as the one shown in  FIG. 3 , the manifold plate and filter plate may be combined into one piece. 
     The biological sample collector may have several interchangeable components that may be readily assembled in field to collect the biological samples. In one embodiment biological sample collector may comprise a kit of multiple manifold plates, filter plates, slide cases and tube cases, as well as different types of housing shapes. Also, slide case may be interchanged with tube case depending on the application. This interchangeability provides a portable solution to biological sample collection that can adapt to a variety of collection situations, thus increasing the flexibility of use. 
     Another advantage of a modular design is that each of the plate and case components may be cleaned to reduce cross-contamination between collections of biological samples. Further modular components allow each plate and case component to be made of a disposable material, thus eliminating cross-contamination between collections of biological samples. 
     The housing may also have controls or a microprocessor (not shown) that operate components of housing. Actuator may be a button, switch, trigger, etc. that controls air pump. Air pump, which acts as a vacuum, sucks air in through conduit. Vent grate provides ventilation for air pump. The air pump may have a motor that operates at approximately 5-30 ft/min. 
     Base extension has one or more clamps for securing manifold plate, filter plate and slide case or tube case mounted thereon. Clamps may be any suitable releasable retention device that keeps the components mounted on base extension when in operation. Examples of suitable retention device include clamps, straps, fasteners, screws, grips, etc. In some embodiments there may separate clamps for each component mounted upon the base extension. 
     Connected to housing through power port may be a power source. Power source may include a battery or wall outlet. For portable applications, a battery may be used. Battery may have an activation switch and/or a clip. Conventional primary and secondary batteries may be used with embodiments of the present invention. 
     In one embodiment, housing is made of a translucent material, such as an acrylic material or other similar plastic material. A translucent material allows the operator of the biological sample collector to visually confirm that all the components are properly mounted. In addition, this allows for inspection of the biological sample deposited during use. 
     The shape and style of biological sample collector may vary. One example of a planar shape is shown in  FIG. 1 . Examples of a gun-shaped housing are shown in  FIGS. 2-4 . The embodiment in  FIG. 4  shows a biological sample collector  400  comprising a gun-shaped housing  402 , manifold plate  404 , filter plate  406 , slide case  408 , actuator  412 , power connector  420 , power source  428 , tube  460  and hollow slider  470 . It should be understood that other housing shapes and styles may be used with embodiments of the present invention. 
     A manifold plate is one of the components mounted on the housing. The configuration of manifold plate may vary depending on the application. For example, there may be a network of pipes that connect the plurality of intake valves and the outlet valve. It should be understood that the number of intake valves may correspond to the design of filter plate. Each intake valve may have an annular sealing ring, such as an O-ring. This annular ring engages the filter well of the filter plate. When manifold plate is mounted to housing the outlet valve aligns with and sealingly engages the exposed orifice of the conduit connected to the air pump. 
     Any suitable filter plate may be used with embodiments of the present invention. The filter plate has a tapered end which sealingly engages with one of the intake valves when mounted to manifold plate. Filter may be made of a PDVF, Telphon™, or similar material. In one embodiment the filter may have a 0.45 μM or 0.8 μM pore size. The pore size should be sufficient to prevent any of the biological sample from entering the manifold pipe or air pump. 
     Tube is flexible and has an opened end that is used to collected biological samples from a treated environment. The open end may be a nozzle or similar vacuum port. 
     The closed mortise-tenon joint between notch and tenon aligns and secures the lid to the housing. This may act to further hold the manifold and filter plates to the housing. In some embodiments the hollow slider is driven by a mechanical motor. Mechanical motor may be within slide case and receives power from the power source through housing. The notch and tenon may also have an electrical mating connection that provides power and control through the housing. Thus, once the actuator on the housing is pressed the air pump may turn on and a control signal may be sent to the hollow slider to move to the next filter well. 
       FIG. 5A  is a perspective view of a slide case  508  comprising a frame lid  562  with an enclosed space  564 , inner walls  566 , and grooves  568 . Grooves  568  are cut into opposing inner walls  566 . Grooves  568  may be inclined at one end of inner wall  566  to allow hollow slider  570  to be inserted into slide case  508 . 
     Within enclosed space  564 , a hollow slider  570 , shown in  FIGS. 5B-5E , travels. Hollow slider  570  has guide rails  572  extending from body  574  through which a cylinder  576  having an empty bore extends. Guide rails  572  fit within grooves  568  and define a travel path of hollow slider  570 . In one embodiment, when tube  560  is inserted into cylinder  576  pinchers  578  may be used to hold tube  560  in place.  FIG. 5D  shows pinchers  578  slid open when inserting tube  560  and closing shut as shown by arrows  580  to grasp tube  560 . When open the bore size of cylinder  576  may increase.  FIG. 5E  shows that when tube  560  is pushed down, pinchers  578  may snap down as shown by arrow  582 . 
     Hollow slider may be moved manually or by a mechanical means such as an electrical motor that drives a gears or wheels. Other mechanisms for moving the hollow slider are contemplated by this invention. In addition, there may be an alignment indicator next to each filter well to confirm the position of the hollow slider. 
     There may be a releasable mechanism for locking the hollow slider in place once aligned, such as the reciprocal teeth shown in  FIG. 2 . Other similar releasable locks may also be used with different embodiments of the present invention. 
     Returning to the embodiment shown in  FIG. 1 , a detailed cross-section view of a tube  160  inserted into trap  144  of a filter well  140  is shown in  FIG. 6 . Biological sample collector  100  has a slide case  108  mounted on filter plate  106  which is mounted on manifold plate  104 . Tube  160  sealingly engages with film barrier  152  when inserted downward into trap  144 . Arrows  186  indicate the direction of the air flow through tube  160 , into filter well  140  and out through pipe  134 . Biological samples (not shown) are deposited on filter  148 . In one embodiment, when tube  160  is pushed down tube  160  slides within cylinder  176  of hollow slider  170 . To create a proper seal, intake valve  138  sealingly engages with tapered end  150  of filter well  140 . The seal between tube  160  and trap  144  is releasable once the biological sample is deposited on filter  148 . 
     A top view of film barrier  752  is shown in  FIG. 7 . Film barrier  752  has several slots  788  for flexible flaps  790 . Although four slots  788  are shown in  FIG. 7 , other embodiments may have a different number of slots from approximately 2-20 slots. Film barrier  752  is placed over the trap of each filter well to prevent aerosolization of any biological sample that is deposited into the filter well. In one embodiment, a plurality of film barriers  752  may be placed on a sheet (not shown) which correspondingly aligns with each of the filter wells. Film barrier  752  may be made of any suitable plastic material. 
       FIGS. 8A and 8B  illustrate two loading positions of hollow slider  170  of the embodiment shown in  FIG. 1  in accordance with an exemplary method of the present invention. Prior to collecting the biological samples, biological sample collector  100  is assembled. Manifold plate  104  is mounted on a base extension  118 , and filter plate  106  is mounted on top of manifold plate  104 . It should be understood that the selection of manifold plate  104  and filter plate  106  is such that the number of filter wells  140  corresponds to the intake valves  138 . Next, slide case  108  is placed over filter plate  106  and housing  102 . Clamp  126  secures each of these plates when mounted to housing  102 . 
     Next, tube  160  is loaded into cylinder  176  of hollow slider  170 . Hollow slider  170  travels along grooves  168  in slide case  108 . Once aligned with a filter well  140 , tube  160  is pushed down through film barrier  152  into trap  144  and air pump  114  is activated by actuator  112 . Power from power source  126  drives the motor of air pump  114 . Air pump  114  sucks the biological sample through tube  160  and deposits the biological samples collected on to filter  148 . Once the biological sample is deposited, the vacuum shuts off and tube  160  is retracted out of the trap  160  through the film barrier  152 . Hollow slider  170  travels along grooves to the next filter well  140 ′ shown in  FIG. 8B  and the process is repeated. This continues until each filter well is loaded with biological samples. Next clamps  126  are released and slide case  108  is removed so that the filter plate  106  may be replaced with an empty filter plate  106 . Each of the loaded filter plates may be assembled into a larger plate for testing of the collected biological sample. 
     All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference. 
     Although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.