Patent Publication Number: US-9423398-B2

Title: Apparatus and method for biologic sample rapid collection and recovery device, and convenient storage

Description:
FIELD OF THE INVENTION 
     This invention relates to apparatus and method for biologic sample rapid collection and recovery device, and convenient storage that includes, but is not limited to, collecting, storing and recovering nucleic acid from a microorganism. 
     BACKGROUND 
     Polymerase chain reaction (“PCR”) is a powerful molecular biology technique used to copy, i.e., amplify, specific nucleic acid sequences within template deoxyribonucleic acid (“DNA”), whether derived from native DNA or cDNA reverse transcribed from ribonucleic acid (“RNA”). An entire PCR assay is carried out in a single tube containing a mixture of enzyme, template, primers, and substrates. Each amplification cycle starts with denaturation (which includes at least heating) that is followed by annealing and then elongation (also known as synthesis and polymerization) reactions. 
     The PCR assay, in which the cycle of denaturing, annealing and synthesizing reactions is typically repeated 20 or more times, can be divided into three phases: exponential when the PCR reaction product doubles during every cycle (assuming 100% reaction efficiency); linear when the reaction components are consumed, and the reaction product growth slows and degrades; and plateau when the reaction product growth stops and degrades. In traditional (also known as End-Point, or classic) PCR, detection and quantitation of the amplified sequence, i.e., the reaction product, are performed in the plateau phase. In real-time PCR, the amount of PCR reaction product is detected and measured at each cycle during the exponential growth phase, which enables determination of the initial amount of DNA template with great precision. 
     Enzyme-linked immunosorbent assay (“ELISA”) is a commonly used diagnostic technique, in which an enzyme is coupled directly to an antibody. The antibody bound to an unknown amount of antigen can be quantitated indirectly by measuring the conversion by the enzyme of an ELISA substrate to a product. A detectable signal can permit real-time determination of the ELISA reaction rate, e.g., a color change can be used to quantify enzyme reaction through photometric adsorbency measurement. 
     Lateral flow technology (“LFT”) assay (also known simply as strip-test) has been a popular platform for diagnostic tests since its introduction in the late 1980s, e.g., the human early pregnancy test. LFT assay has been used for qualitative or semi-quantitative detection of specific analytes including antigens and antibodies, hence other common names for LFT assay: lateral flow immunoassay and immunochromatographic strip test. Biologic sample for LFT assay may be derived from whole blood, serum, plasma, saliva, urine, feces, wound exudate, soil, dust, vegetation, food, or other suitable source. Even products of nucleic acid amplification systems, such as PCR reaction products, can be studied by LFT assay. LFT assay can be run in a single step using the biologic sample in a variety of test locations, such as laboratory, agricultural field, crime scene, etc. Several analytes can be tested simultaneously on the same strip. 
     The prior art requires a burdensome and time consuming step of eluting (also known as extracting) an analyte from an LFT device before performing the PCR or ELISA technique on the analyte collected by LFT assay. There exists a need in the art for an LFT device configured for collecting (also known as sampling, fixing, or binding) the analyte that may be placed directly in an analysis system for analysis, such as by the PCR or ELISA technique, without the eluting step intervening. Eliminating the eluting step would speed and simplify the collection of specimens for molecular-clinical diagnostics. The apparatus and method embodied by the claims finds application when sampling specimens for molecular-clinical diagnostics in human health, veterinary health, plant health, biosecurity (e.g., surveillance and response), defense, forensics, microbial forensics, and food quality and biosecurity, among other fields of study. 
     SUMMARY OF THE INVENTION 
     In accordance with exemplary embodiments, apparatus and method for biologic sample rapid collection and recovery device, and convenient storage are provided. 
     An exemplary embodiment includes an apparatus comprising a lateral flow technology (“LFT”) device including at least a membrane configured to bind an analyte from a sample that flows through the LFT device, in which a selected portion of the membrane bound to the analyte when placed directly in an analysis system does not substantially inhibit analysis of the analyte. 
     An alternative exemplary embodiment includes an apparatus comprising an LFT system including at least a membrane configured to bind an analyte from a sample that flows through the LFT system, such that the LFT system facilitates a period of storage of the analyte bound to the membrane and does not substantially inhibit analysis of the analyte bound to the membrane when placed in an analysis system after the period of storage. 
     Another exemplary embodiment includes a method comprising, the steps of: flowing a sample solution that contains an analyte through an LFT device configured to bind the analyte; punching a sample disc that binds the analyte from the LFT device; and placing the sample disc directly in an analysis system for analysis of the analyte, in which the step of placing the sample disc is not preceded by a step of eluting the analyte from the sample disc. 
     These and various other features and advantages that characterize the claimed invention will be apparent upon reading the following detailed description and upon review of the associated drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows plan view of an exemplary lateral flow technology device. 
         FIG. 1A  illustrates a sectional view taken along the line  1 A- 1 A of  FIG. 1 . 
         FIG. 2  shows plan view of another exemplary lateral flow technology device. 
         FIG. 2A  illustrates a sectional view taken along the line  2 A- 2 A of  FIG. 2 . 
         FIG. 3  shows plan view of an alternate exemplary lateral flow technology device. 
         FIG. 3A  illustrates a sectional view taken along the line  3 A- 3 A of  FIG. 3 . 
         FIG. 4A  portrays a plan view of a portion of an exemplary direct format lateral flow technology assay before the assay is run. 
         FIG. 4B  depicts a plan view of a portion of the exemplary direct format lateral flow technology assay after the assay is run and the result is positive. 
         FIG. 4C  reveals a plan view of a portion of the exemplary direct format lateral flow technology assay after the assay is run and the result is negative. 
         FIG. 5A  portrays a plan view of a portion of an alternate exemplary competitive format lateral flow technology assay before the assay is run. 
         FIG. 5B  depicts a plan view of a portion of the alternate exemplary competitive format lateral flow technology assay after the assay is run and the result is negative. 
         FIG. 5C  reveals a plan view of a portion of the alternate exemplary competitive format lateral flow technology assay after the assay is run and the result is positive. 
         FIG. 6  shows an exemplary embodiment where a selected portion of the membrane is placed directly in a PCR test tube of an analysis system. 
         FIG. 7  shows an exploded view of an exemplary lateral flow technology device with an optional cassette. 
         FIG. 8  shows an SEM picture of bacteria on water soluble paper. 
         FIG. 9  is a graphic representation of absorbance readings for different water soluble papers. 
         FIG. 10  is a graphical representation of absorbance readings for different amounts of water. 
         FIG. 11  is a graphical representation of absorption times for different water soluble papers. 
         FIG. 12  is a graphical representation of absorption time for different amounts of water. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Detailed descriptions of the exemplary embodiments are provided herein. It is to be understood, however, that the invention embodied in the claims may take various forms. Various aspects of the invention may be inverted, or changed in reference to specific part shape and detail, part location, or part composition. In addition, the figures shown are not drawn to scale. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention embodied in the claims in virtually any appropriately detailed system, structure or manner. 
     OVERVIEW 
       FIG. 1  shows an exemplary configuration of a lateral flow technology (“LFT”) device  10  that includes at least a sample pad  12 , a membrane  14 , and a wick  16 .  FIG. 1A  sectional view depicts the sample pad  12 , the membrane  14 , and the wick  16  overlap one another and are mounted on a backing card  20 , such as with an adhesive  22 , e.g., a pressure-sensitive adhesive, therebetween. In one embodiment a MIBA-020 backing card with an adhesive coating is used. As discussed below, it has been found that an unmounted portion  24  of the membrane  14  produces useful characteristics. 
     An LFT assay is run by bringing a sample  26  in contact, e.g., in a sample application direction  28 , with the sample pad  12  of the LFT device  10 . The sample  26  flows (in a flow direction  34 ) through the sample pad  12  into the membrane  14 . Some portion of an analyte  32  in the sample  26  may become fixed in the membrane  14 . The sample  26 , including the analyte  32 , not fixed to the membrane  14  flow (in the flow direction  34 ) towards the wick  16  where the sample  26 , including the analyte  32 , are entrapped (also known as absorbed). In certain embodiments, the sample  26  is a biologic sample and the analyte  32  is nucleic acid from a microorganism. 
       FIG. 2  shows another exemplary embodiment of the LFT device  10  with a conjugate pad  18  disposed between the sample pad  12  and the membrane  14 . As illustrated in the  FIG. 2A  sectional view, the LFT assay is run by bringing the sample  26  in contact, e.g., in the sample application direction  28 , with the sample pad  12  of the LFT device  10 . The sample  26  flows (in the flow direction  34 ) through the sample pad  12  to the conjugate pad  18 , where the particulate conjugate  30  has been immobilized. The sample  26  mobilizes the particulate conjugate  30 , and the analyte  32  in the sample  26  interacts with the particulate conjugate  30  as they flow (in the flow direction  34 ) into the membrane  14 . Some portion of the particulate conjugate  30  and the analyte  32  may become fixed in the membrane  14 . The excess of the sample  26 , including the analyte  32 , and the particulate conjugate  30  not fixed to the membrane  14  flow (in the flow direction  34 ) towards the wick  16  where they are entrapped. The conjugate pad  30  is optional. 
       FIG. 3  portrays an alternative embodiment of the LFT device  10 , in which a specimen filter  36  is disposed between the sample pad  12  and the membrane  14 . As shown in the  FIG. 3A  sectional view, the specimen filter  36  is configured to collect a sample debris  38  from the sample  26  while allowing the analyte  32  to flow (in the flow direction  34 ) from the sample pad  12  through the specimen filter  36  to the membrane  14  and into the wick  16  when the sample  26  is brought in contact, e.g., in the sample application direction  28 , with the sample pad  12  of the LFT device  10 . The absorbed fluid of the sample  26  flows (in the flow direction  34 ) through the specimen filter  36  to help separate the specimen fractions, e.g., the analyte  32 , and cell debris, e.g., the sample debris  38 , by size. The specimen filter  36  is optional. 
       FIGS. 1-3A  demonstrate various embodiments; however, it will be understood that alternative embodiments can be utilized, e.g., any embodiment may or may not have the unmounted portion  24  of the membrane  14 . Similarly, any of the various embodiments can have the holder  46  (as shown in  FIGS. 2-2A ) configured for handling the LFT device  10  or providing a label space for the LFT device  10 . 
     Lateral Flow Technology Assay Formats 
     The LFT device  10  can further include either direct (also known as sandwich technique), competitive (also known as inhibition technique), or “boulders in the stream” format LFT assay, as readily known to those skilled in the art. However, these assay formats are optional. 
       FIGS. 4A-C  illustrate a portion of the LFT device  10  showing the conjugate pad  18  and the membrane  14 .  FIG. 4A  represents the LFT device  10  before the direct format LFT assay is run.  FIG. 4B  shows the LFT device  10  after the assay is run (in the flow direction  34 ) for a positive result. A test line  40  is visible (depicted as black) and a control line  42  is visible.  FIG. 4C  portrays the LFT device  10  after the assay is run (in the flow direction  34 ) for a negative result. The test line  40  is invisible (depicted as bounded by broken lines) and the control line  42  is visible. 
       FIGS. 5A-C  illustrate a portion of the LFT device  10  showing the conjugate pad  18  and the membrane  14 .  FIG. 5A  represents the LFT device  10  before the competitive format LFT assay is run.  FIG. 5B  shows the LFT device  10  after the assay is run (in the flow direction  34 ) for a negative result. The test line  40  is visible and the control line  42  is visible.  FIG. 5C  portrays the LFT device  10  after an assay is run (in the flow direction  34 ) for a positive result. The test line  40  is invisible and the control line  42  is visible. 
     Further details regarding LFT technology are given below, and the book  Lateral Flow Immunoassay  edited by Raphael Wong and Harley Tse, Humana Press (2009) provides further information and is incorporated by reference herein. 
     Membrane 
     One purpose of the membrane  14  (also known as analytical region, reaction membrane, or matrix membrane) in the LFT device  10  is to bind the analyte  32 , such as protein, antigen, antibody, nucleic acid, or microorganism. 
     Traditionally, nitrocellulose has been the material of choice for the membrane  14  in the vast majority of LFT devices  10 . Other traditional materials for the membrane  14  have included nylon and polyvinylidene fluoride. Nitrocellulose has flaws, such as flammability and breakage problems when the membrane  14  does not adhere to the backing card  20 . The traditional materials have shelf-life longevity issues. In addition, the analyte  32  sampled by the LFT device  10  must be extracted from the membrane  14  made of these traditional materials (nitrocellulose, nylon, polyvinylidene fluoride, etc.) by the burdensome and time-consuming process of elution in order for the analyte  32  to be analyzed in an analysis system, such as those using the PCR or ELISA technique, such as is described in (I) IUPAC Gold Book: eluent. International Union of Pure and Applied Chemistry, www.goldbook.iupac.org/E02040.html. Retrieved 2008-09-28; (2) Brown, Phillis (2001). Advances in chromatography. CRC Press. pp. 36. ISBN 082470509; and (3) Elution. www.en.wikipedia.org/wiki/Eiution; each of which are hereby incorporated by reference. 
     However, it has been found that by selecting an appropriate material that does not substantially inhibit (or interfere with or impede) PCR, the membrane  14  may be placed directly into End-Point PCR mixtures, reverse transcription PCR mixtures, real-time PCR mixtures, without the eluting step intervening and variants of these methods. Alternatively, the membrane  14  may be placed directly, without the eluting step intervening, in an analysis system using the ELISA technique. In other words, the membrane  14  collects the analyte  32  in the LFT device  10  when the LFT assay is run, and the membrane  14  acts a carrier of the analyte  32  into the PCR or ELISA system, or other suitable analysis system. 
     As shown in  FIG. 6 , after the LFT assay has been run (in the flow direction  34 ) from the sample pad  12  to the wick  16 , a membrane disc (also known as a “punch” or “selected portion” or “sample disc”)  48  is obtained from the membrane  14  made of a suitable biomaterial in an exemplary embodiment. The punch  48  is a selected portion of the membrane  14 , e.g., 1.2 millimeter (“mm”) diameter piece obtained with a Whatman® Harris Uni-Core™ 1.2 mm (“puncher”), which is placed directly into a PCR tube (depicted as  50 ) or an ELISA microplate well of the PCR or ELISA analysis system, respectively. Other suitable sizes and shapes for the punch  48  may be used in various embodiments. 
     In a particular exemplary embodiment, the membrane  14  is made from a water soluble paper, e.g., Aquasol® Water Soluble Paper is a unique product made of sodium carboxy methyl cellulose (“SCMC”) and wooden pulp that dissolves in cold water, hot water, steam and most aqueous solutions. In addition, Aquasol® Water Soluble Paper is described as non-toxic, environmentally friendly, and 100% biodegradable. Aquasol® Water Soluble Paper can be obtained from Aquasol Corporation (North Tonawanda, N.Y. USA). 
     Water soluble paper containing SCMC is available in a variety of SCMC cards that can be used to make the membrane  14 . SCMC cards evaluated for the membrane  14  are ASW-15, which has 50 μm thickness; ASW-25, which has 83 μm thickness; ASW-50, which has 76 μm thickness; ASW-DC, which has 50 μm thickness; and ASW-240, which has 170 μm thickness. However, other material with suitable characteristics disclosed may be utilized for the membrane  14 . The more preferred SCMC cards from the above five SCMC cards are ASW-15, ASW-25 and ASW-50, because these SCMC cards did not substantially inhibit PCR up to a fifth order of magnitude dilution of a test using plasmid-fungal DNA. The starting solution is diluted four times for a total of five concentrations, i.e., A=10 ng/μl; B=1 ng/μl; C=0.1 ng/μl; D=0.01 ng/μl; E=0.001 ng/μl; F=is negative control, water only no DNA. 
     Conjugate Pad 
     The role of the conjugate pad  18  (as shown in  FIGS. 2-2A ) in the LFT device  10  includes accepting the particulate conjugate  30 , holding the particulate conjugate  30  stable over shelf life (preferably up to 2 years) of the LFT device  10 , and releasing the particulate conjugate  30  efficiently and reproducibly when the LFT assay is run. As the skilled artisan will know, it is often necessary to pretreat the conjugate pad  18  to assure optimal release and stability of the particulate conjugate  30 . 
     Materials of choice for the conjugate pad  18  are glass fiber, polyester or Rayon. In an exemplary embodiment, the conjugate pad  18  is made from nitrocellulose. For the best results, the materials for the conjugate pad  18  should be hydrophilic and allow rapid flow rates. 
     The particulate conjugate  30  may be adsorbed with antibodies or antigens that are specific to the analyte  32  to be collected. A label for the particulate conjugate  30  may include colloidal gold or monodisperse latex, tagged with either a visual or a fluorescent dye. The labels can be read qualitatively or quantitatively after the assay is run. 
     As previously mentioned, the conjugate pad is optional. 
     Sample Pad 
     The role of the sample pad  12  (e.g., as shown in  FIGS. 1-3A and 6 ) in the LFT device  10  includes accepting the sample  26 , treating the sample  26  such that it is compatible with the LFT device  10 , and releasing with high-efficiency the analyte  32  into the rest of the LFT device  10 , e.g. the membrane  14 . The sample pad  12  must be able to accept in a controlled way all of the fluid volume from the sample  26 , thereby helping to channel the fluid into the assay materials (such as the conjugate pad  18 , the membrane  14 , and the wick  16 ) rather than allowing flooding or surface flow. Sample treatments for the sample pad  12  include filtering unwanted particulates, changing the pH, binding sample components that can inhibit running the LFT assay, and disrupting sample components, such as mucins, to release the analyte  32  to the LFT device  10 . In some embodiments, the sample pad  12  and the conjugate pad  18  can be the same component. 
     Tensile strength while the sample pad  12  is wet from the sample  26  is an important criterion used in selecting the material for the sample pad  12 . In an exemplary embodiment, the fluid volume of the sample  26  used to run the LFT assay is 0.5 milliliters (“ml”), or 500 microliters (“μl”). 
     Examples of materials used for the sample pad  12  are cellulose, glass fiber, Rayon, and other filtration media. In exemplary embodiments, the sample pad  12  is made from Whatman GB002, Standard 17, or GF33 (from Whatman plc, UK); or Millipore C083 (from Millipore in Billerica, Mass. USA). 
     Wick 
     The wick  16  (e.g., as shown in  FIGS. 1-3A and 6 ) acts as the engine of the LFT device  10  while running the LFT assay to pull fluid from the sample  26  added to the LFT device  10  into the wick  16  and hold the fluid for the duration of the assay and in an exemplary embodiment for the duration of storage until recovery of the analyte  32  by the punch  48  from the membrane  14 . The wick  16  should not release fluid back into the membrane  14  or false positive results may occur. 
     The wick  16  is typically made from high-density cellulose, which is generally selected for absorptive capacity and tensile strength. An exemplary wick  16  is made from Millipore C083. 
     “Wicking rate” or “capillary rise time” is defined as the time required for a fluid front of the sample  26  to traverse the distance from the sample pad  12  through the membrane  14  to absorption at the wick  16 . Wicking rate is an important criterion when selecting the materials for making the sample pad  12 , the membrane  14 , the wick  16 , etc. In an exemplary embodiment, the wicking rate is chosen to be less than the time for the membrane  14  made from water soluble paper to dissolve in the fluid of the sample  26 . 
     Specimen Filter 
     The role of the specimen filter  36  (as shown in  FIGS. 3-3A ) is separation of the specimen fractions, e.g., the analyte  32 , and the cell debris, e.g., the sample debris  38 . The specimen filter may be made from a variety of materials known to one skilled in the art. In an exemplary embodiment, the specimen filter  36  is made from nitrocellulose. 
     The specimen filter  36  is optional. The sample pad  12  may provide some of the filtration function that could be provided by the specimen filter  36 . 
     Backing Card 
     The role of the backing card  20  (as shown in  FIGS. 1A, 2A, and 3A ) includes providing rigidity to the LFT device  10 , which eases handling of the LFT device. The backing card  20  can be made from polystyrene or other plastics, or other suitable material. In an exemplary embodiment, the backing card  20  is made from Part # MIBA-020 obtained from Diagnostic Consulting Network (Carlsbad, Calif. USA). 
     Next, actual examples of the LFT device and components are described below. 
     Exemplary Lateral Flow Device 
       FIG. 7  portrays an exemplary embodiment in exploded view of the LFT device  10  surrounded by a cassette (also known herein as “housing”)  58 . The sample pad  12  and wick  16  are made from Millilpore C083. The membrane  14  is made from Aquasol ASW-25. The backing card  20  is made from MIBA-020. The fluid volume of the sample  26  used for the exemplary embodiment is 0.5 ml. The wicking rate is approximately 3 minutes, i.e., 2.94 minutes, as measured by the mean of ten test runs of the sample  26  through the LFT device  10  for the time required for a fluid front of the sample  26  to traverse from the bottom of the sample pad  12  to the top of specimen filter, i.e., a 50 mm travel distance. The average speed is 17 mm/min. 
     In the exemplary embodiment, the plastic cassette  58  is MICA-0120 sold by Diagnostic Consulting Network (Carlsbad, Calif.), herein incorporated by reference. The outside length  60  of the plastic cassette  58  is 69 mm. The outside width  62  of the plastic cassette  58  is a cassette width of 20 mm. The cassette  58  is optional. 
     In the exemplary embodiment, the cassette  58  has an upper housing member  64  and a lower housing member  66 . The upper housing member  64  has an attachment element  68  (shown in partial cutaway) and correspondingly the lower housing member  66  has an engagement element  70 . The attachment element  68  and the engagement member  70  may be reversed between the upper housing member  64  and the lower housing member  66 . The attachment element  68  can reversibly engage the engagement element  70  for containment of the LFT device  10  by the housing  58 . The upper housing member  64  and the lower housing member  66  may have a plurality of attachment elements  68  and engagement elements  70 , respectively. 
     The upper housing member  64  has a well  72  for receiving fluid from the sample  26  and a well aperture  74  at the base of the well  72  for releasing fluid into the sample pad  12 . The well  72  may receive 0.5 ml of fluid without overflowing. A rim  76  of the well aperture  74  may be biased into the sample  12  when the cassette  58  contains the LFT device  10  so that fluid from the sample  26  does not flood or surface flow the LFT device  10 . 
     The upper housing member  64  has a membrane window  78  through which LFT assay results may be viewed without opening the cassette  58  containing the LFT device  10 . In addition, the punch  48  from the membrane  14  can be obtained without opening the cassette  58 . In the MIBA-0120, S (labeled  80 ) for the sample  26 , T (labeled  82 ) for the test line  40 , and C (labeled  84 ) for the control line  42  are provided. A gripping area  86  is provided, also. 
     The lower housing member  66  has a sample guide  88  that facilitates biasing the sample pad  12  towards the well aperture  74  when the LFT device  10  is contained within the cassette  58 . The pedestal  90  facilitates biasing the membrane  14  towards the membrane window  78  when the LFT device  10  is contained within the cassette  58 , which promotes efficiently obtaining the punch  48 . A wick guide  92  provides lateral support to the LFT device  10  adjacent the wick  16  to help maintain positions of the sample pad  12  and the membrane  14  relative to the well aperture  74  and the membrane window  78 , respectively. 
     In the exemplary embodiment of the LFT device  10 , the width  94  of each of the sample pad  12 , the membrane  14 , the wick  16 , and the backing card  20  is 4 mm; the length  96  of the sample pad  12  is 26 mm; the length  98  of the membrane  14  is 25 mm; the length  100  of the wick  16  is 21 mm; and the length  110  of the backing card  20  is 67 mm. The sample pad  12  has an unmounted portion  102  that overlaps the membrane  14  by 2.5 mm at the first end  104  of the membrane  14 . The remaining 23.5 mm of the sample pad  12  is mounted to the backing card  20  by the adhesive  22 . The wick  16  has an unmounted portion  106  that overlaps the membrane  14  by 2.5 mm at the second end  108  of the membrane  14 . The remaining 18.5 mm of the wick  16  is mounted to the backing card  20  by the adhesive  22 . The first end  104  and the second  108  are mounted to the backing card  20  by the adhesive  22 , while the unmounted portion  24 , which in this exemplary embodiment has a length of 20 mm, of the membrane  14  is not adherent to the backing card  20 . 
     The unmounted portion  24  facilitates obtaining the punch  48  (as shown in  FIG. 6 ) of the membrane  14  without the backing card  20  (shown in  FIG. 6 ) adherent. The punch  48  thus obtained may be placed directly in the analysis system for analysis of the analyte  32  without first needing to elute the analyte  32  from the punch  48 , when the backing card  20  adherent to the punch  48  could interfere with analysis of the analyte  32 . 
     When the plurality of attachment members  68  of the upper housing member  64  are correspondingly coupled to the plurality of the engagement members  70  of the lower housing member  66  upon closing the plastic cassette  58 , the LFT device  10  is held relatively immobile in a protective container that promotes convenient storage. In preferred embodiments, the LFT device  10  conveniently stores the analyte  32  collected on the membrane  14  for at least 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months at room temperature (˜23° C.), in a refrigerator (˜4° C.), in a freezer (˜−20° C.), or in a deep freezer (˜−80° C.), or in some combination of temperatures. To present positive storage has been determined for eight months for types of soluble membrane at all temperatures. Considering the observed good yields of DNA it is projected at least 24 months of storage. After the convenient storage, the analyte  32  is reliably recovered by the punch  48  and carried to the analysis where the analyte  32  is analyzed in the analysis system. 
     In other words, an exemplary embodiment of an apparatus has an LFT system including at least a membrane  14  configured to bind an analyte  32  from a sample  26  that flows through the LFT system, such that the LFT system facilitates a period of storage of the analyte  32  bound to the membrane  14  and does not substantially inhibit analysis of the analyte  32  bound to the membrane  14  when placed in an analysis system after the period of storage. A second embodiment includes a selected portion  48  of the membrane  14  is water soluble paper placed directly in the analysis system for analysis of the analyte  32 . A third embodiment includes the period of storage is substantially at a predetermined temperature chosen from a group consisting of room temperature, refrigeration, freezing, and deep freezing. A fourth embodiment includes the period of storage is at least 3 months before the selected portion  48  is placed directly in the analysis system, and the analysis system includes at least an analysis technique selected from a group consisting of PCR and ELISA. 
     Punch from Water Soluble Paper 
     It has been found that the appropriately selected membrane  14  can capture the analyte  32  of interest, yet still present the analyte  32  for analysis in the analysis system while not substantially inhibiting the analytic technique. An exemplary embodiment of such a membrane  14  is water soluble paper. Various water soluble papers with different thicknesses were tested. It was found that the thickest material has more residue as seen by higher absorption reading. In addition, the thickest material absorbed water faster than the thinnest material. The application of water produced no perforation, fissures, cracking, holes or breakage in the water soluble paper even with the test water volume increased to 10 μl. 
     A scanning electron microscope (“SEM”) was used to determine the surface appearance of water soluble paper in the previously wet versus dry states, and to assess water soluble paper looking for the presence of pore spaces, or crevices, that may serve as storage pockets for the analyte  32 , such as a microorganism or nucleic acid. A small piece of water soluble paper (dry versus previously wet by 10 μL water then allowed to dry) was treated with gold-palladium coating and viewed under SEM at magnification=100×, Spot=3.0, and HV=10.00 kV. It was found that the organization of the paper fibers in the previously wet water soluble paper is more scattered than in the dry water soluble paper. 
     The water soluble paper was studied for the ability to bind (or fix) bacteria, i.e., analyte  32 , to the soluble paper using SEM analysis. Due to the solubility properties of water soluble paper, fixation of bacteria  32  in water soluble paper had some challenges. However, bacteria  32  could be trapped in the water soluble paper, particularly in the crevices (see  FIG. 8 ). 
     An embodiment includes a storage crevice of the membrane  14  configured to bind the analyte  32 . A second embodiment includes the storage crevice is absent before the sample flows through the membrane  14 . 
     In a particular exemplary embodiment, the membrane  14  is made from a water soluble paper, e.g., Aquasol® Water Soluble Paper is a unique product made of sodium carboxy methyl cellulose (“SCMC”) and wooden pulp that dissolves in cold water, hot water, steam and most aqueous solutions. Water soluble paper containing SCMC is available in a variety of SCMC cards that can be used to make the membrane  14 . SCMC cards evaluated for the membrane  14  are ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240. However, other material with suitable characteristics disclosed may be utilized for the membrane  14 . The more preferred SCMC cards from the above five SCMC cards are ASW-15, ASW-25 and ASW-50, because these SCMC cards did not substantially inhibit PCR up to a fifth order of magnitude dilution of a test using plasmid-fungal DNA. The starting solution is diluted four times for a total of five concentrations, i.e., A=10 ng/μl; B=1 ng/μl; C=0.1 ng/μl; D=0.01 ng/μl; E=0.001 ng/μl; F=is negative control, water only, no DNA. 
     The punch  48  taken from the unmounted portion  24  of membrane  14  (as also seen in  FIG. 1A ) can be placed directly into the analysis system when the membrane  14  is made from material that does not interfere with the analysis technique, such as the PCR or ELISA technique. Thus, the exemplary embodiment yields a robust apparatus and method for biologic sample that reduces, and may eliminate, the need of tedious extraction protocols for the analyte  32 . 
     In other words, an exemplary embodiment is an apparatus having an LFT device  10  including at least a membrane  14  configured to bind an analyte  32  from a sample  26  that flows through the LFT device  10 , in which a selected portion  48  of the membrane  14  bound to the analyte  32  when placed directly in an analysis system does not substantially inhibit analysis of the analyte  32 . A second embodiment includes an elution protocol is not required to extract the analyte  32  bound from the selected portion  48  before placed directly in the analysis system. A third embodiment includes the selected portion  48  of the membrane  14  is water soluble paper. A fourth embodiment includes the analysis system includes at least an analysis technique selected from a group consisting of PCR and ELISA. A fifth embodiment includes the selected portion  48  of the membrane  14  includes at least sodium carboxy methyl cellulose. A sixth embodiment includes the selected portion  48  of the membrane  14  bound to the analyte  32  is placed directly in the analysis system without elution of the analyte  32  from the membrane  14 , and the analysis system includes at least an analysis technique selected from a group consisting of PCR and ELISA. 
     In another embodiment, the LFT device  10  further comprises at least a backing card  20  adherent to a mounted portion (such as first  104  or second end  108 ) of the membrane  14 , in which the membrane  14  includes the mounted portions ( 104 ,  108 ) and an unmounted portion  24  that is not adherent to the backing card  20 , and the selected portion  48  is selected from the unmounted portion  24  of the membrane  14 . 
     Analyte Collection 
     In an exemplary embodiment of the LFT device  10 , running the LFT assay collects a portion of the analyte  32 , e.g., microorganism, from the sample  26  in the membrane  14 . Immobilizing specific antibodies to the analyte  32  in the LFT device  10  can increase the analyte  32  collected. Antibodies can be immobilized in a number of combinations on either the conjugate pad  18  or the membrane  14 , which can satisfy current and/or future technical requirements, applications or special demands of the market. 
     To increase the likelihood for a true positive result, rather than a false negative, by the analysis technique, such as PCR or ELISA, the punch  48  is obtained from a high yield area of the membrane  14 . The high yield areas for the punch  48  include the membrane  14  in the test line  40  (as seen in  FIGS. 4B and 5C ) that is positive by immunoassay. 
     In other words, another embodiment includes the LFT device  10  further includes at least a biologic marker, in which the biologic marker is configured to concentrate the analyte  32  in a predetermined sample collection region (such as the test line  40  that is positive, or other biologic marker location) by an assay format chosen from a group consisting of direct, competitive, and boulder in the stream. Another embodiment can include the biologic marker is a specific antibody targeted to the analyte  32 . 
     Another high yield area of the membrane  14  for the punch  48  is a first portion  52  of the unmounted portion  24  of the membrane  14 , as seen in  FIG. 6 . The best location to take the punch  48  is the first 2 mm of the membrane closer to the sample pad  12 . The sample  26  becomes more filtered by sampling within the membrane  14  closer to the wick  16 . In an exemplary embodiment, the first portion  52  is the first 5 mm of the unmounted portion  24  closest to the sample pad  12 , when the unmounted portion  24  length (identified as  54 ) is 20 mm and width (identified as  56 ) is 4 mm. In a further exemplary embodiment, the membrane  14  is sampled within the first 2 mm of the unmounted portion  24  nearest the sample pad  12 . 
     Method for Analysis of Analyte Including Lateral Flow Technology Device 
     An LFT device used in a method for analysis of the analyte  32  is provided. An exemplary method for preparing the sample  26  has at least the steps of grinding a plant tissue sample, performing the LFT assay with the LFT device  10 , and taking a sample disc  48  from the LFT device  10  for use in an analytic system. Then, the step of analyzing the analyte  32  in the analysis system is performed. 
     The preparation of the sample  26  can be performed with the following materials:
         1×PBST (Phosphate buffer saline tween-20);   Sample mesh bag (Agdia ACC 00930 or alternative);   Tissue Homogenizer (Agdia ACC 00900 or alternative);   the LFT device  10 ;   1.5 ml Eppendorf PCR tube;   and the puncher (e.g., Whatman® Harris Uni-Core™ 1.2 mm) for obtaining the punch  48 .       

     The step of grinding of the plant tissue can be performed by the following steps:
         1. Determine the exact amount of plant tissue and 1×PBST buffer needed. 1 g of plant tissue requires 10 ml of 1×PBST. Thus, 0.5 g plant tissue needs 5 ml of 1×PBST.   2. Place the required volume of 1×PBST into a sample mesh bag (Agdia ACC 00930 or alternative).   3. Add the measured amount of plant tissue inside the sample mesh bag containing the 1×PBST. Make sure the plant tissue is between the mesh inside the bag and located near the bottom of the bag.   4. Slightly fold the opening of the bag to prevent spilling while grinding the plant tissue inside the bag using a tissue homogenizer (Agdia ACC 00900 or alternative).   5. Grind the plant tissue until the tissue is completely macerated and the extracted solution becomes translucent.       

     The step of performing the LFT assay with the LFT device  10  can be performed with the cassette  58  or without the cassette  58 . When using the LFT device  10  without the cassette  58 , pipette 0.5 ml of the extracted solution into a 1.5 ml Eppendorf tube. Place the sample pad  12  of the LFT device  10  in the extracted solution in the Eppendorf test tube and allow the solution to move upward until it reaches the wick (˜3 minutes.). After the flow migration, remove the LFT device  10  from the Eppendorf tube. Subsequently, take the punch  48  from the membrane  14  of the LFT device  10 . Subsequently, either obtain a 1.2 mm sample disc  48  or allow the membrane to dry in a clean Petri dish for 3 to 10 minutes. This 3 to 10 minutes drying is optional because the assay works well in both ways wet or dry. However, when the soluble paper is dry it might be easier to punch and manipulate and some user would prefer to wait, but it works well in both ways. 
     When using the LFT device  10  inside the cassette  58 , pipette 0.5 ml of the extracted solution into the well  72  of the cassette  58 , then allow the extracted solution to flow towards the wick (˜3 minutes). Subsequently, take the punch  48  from the membrane  14  of the LFT device  10 . Subsequently, either obtain a 1.2 mm sample disc  48  or allow the membrane to dry in a clean Petri dish for 3 to 10 minutes. 
     The step of taking sample disc  48  from the LFT device  10  for analysis, such as by the PCR or ELISA technique, can be accomplished by the following steps:
         1. Take 1.2 mm sample disc  48  from the membrane  14  by punching it out using a puncher (e.g., Whatman® Harris Uni-Core™ 1.2 mm or alternative).   2. Place the punch  48  directly into a tube for PCR or a ELISA microplate well.       

     Then, the PCR or ELISA based assay is performed according to any appropriate PCR or ELISA protocol. 
     Exemplary Water Soluble Biomaterial 
     A series of experiments were carried out to assess the physical properties of a variety of water soluble biomaterial (“WSB”) that can be used for the membrane  14  in the LFT device  10 . The WSB used for the experiments were obtained from Aquasol® Corporation (North Tonawanda, N.Y.). The WSB are identified by Aquasol® as ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240. The thickness of ASW-15 is 50 μm. The thickness of ASW-25 is 83 μm. The thickness of ASW-50 is 76 μm. The thickness of ASW-DC is 50 μm. The thickness of ASW-240 is 170 μm. 
     In Experiment 1, 10 μl, 15 μl, 20 μl, &amp; 50 μl of water were placed on WSB. An ANOVA (Factor 1: WSB and Factor 2: amount of water) and Duncan&#39;s Multiple Range Test for absorbance readings at 260 nanometers (“nm”) and 280 nm was performed. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. Water was used as control. The amount of water used was 10 μl, 15 μl, 20 μl, and 50 μl. The thickest material had more residues as measured by the highest absorbance, as shown in  FIGS. 9 and 10 . 
     In Experiment 2, a predetermined amount of water was applied to a predetermined amount of WSB. 2, 5, and 10 μl of water were placed on WSB surface. An ANOVA (Factor 1: WSB and Factor 2: amount of water) and Duncan&#39;s Multiple Range Test for absorption time was performed. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. The amount of water used was 2 μl, 5 μl, and 10 μl. The thickest WSB absorbed water faster than the thinnest WSB and the more water was applied to the WSB the slower the applied water was absorbed, as shown in  FIGS. 11 and 12 . 
     In Experiment 3, a predetermined amount of water was applied to a WSB. An ANOVA (Factor 1: WSB and Factor 2: amount of water) and Duncan&#39;s Multiple Range Test for water spot diameter was performed. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. The amount of water used was 2 μl, 5 μl, and 10 μl. The more water was applied to the WSB the larger was the spot. No holes were made in the WSB with any of the water volumes tested wherein “holes” is defined as a perforation, fissure, crack or fiber gap visible to the naked eye. 
     In Experiment 4, the objective was to determine surface appearance, i.e., changes in a matrix of porosity, of WSB and compare “dry” WSB versus “wet” WSB; and to assess WSB for the presence of pore spaces that may serve as storage pockets for the analyte  32 , such as microorganisms and nucleic acids. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. For the “dry” WSB, a small piece of WSB was selected and treated with Gold/Palladium (Au/Pd) coating as known to one skilled in the art. For the “wet” WSB, 10 μl of H 2 O was applied to the WSB and allowed to dry. Then, a small piece of WSB was selected and treated with Gold/Palladium (Au/Pd) coating as known to one skilled in the art. The samples of membranes used for SEM analysis were of square pieces of 13 mm×13 mm. Quanta 600F Field Emission Gun Scanning Electron Microscope (“SEM”) (Hillsboro, Oreg.) was used to analyze the “dry” WSB and “wet” WSB at magnification=100×, Spot=3.0, and HV 10.00 kV. Under SEM, the organization of the fibers in the “wet” WSB is more scattered than in the “dry” WSB, and wetting the WSB caused changes in the physical structure of the WSB such that potential storage pockets are present. Measure of porous size were done and there were not dramatic changes after wetting. 
     Fixation of Analytes in Water Soluble Biomaterial 
     In Experiment 5, the objective was to fix an analyte  32  in a storage pocket in the WSB and view the fixed analyte and the storage pocket under SEM. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. The exemplary analyte  32  was a bacteria, specifically  Pseudomonas syringae  pv. tomato DC3000. 
     The exemplary method steps of a process for a protocol used for fixation of the bacteria to the WSB is commenced at step  5 A by treating a WSB with 10 μl of a bacterial culture of  Pseudomonas syringae  pv. tomato DC3000. 
     In common practice by one skilled in the art, a fixation procedure for the analyte  32  includes soaking the analyte  32  and the membrane  14  in a series of reagents; however, the WSB is soluble so “dropping” 25 μl of the reagents was done instead. 
     At step  5 B, dropping 25 μl of 2% glutaraldehyde onto the WSB treated area acts as a primary fixation, and the glutaraldehyde was let stand for 45 minutes. 
     At step  5 C, dropping 25 μl of 0.1M phosphate buffer onto the WSB treated area cleans the treated area, and the phosphate buffer was let stand for 20 minutes. Process step  5 C is repeated before moving onto process step  5 D. 
     At step  5 D, dropping 25 μl of 1% OsO 4  solution onto the WSB treated area acts as a secondary fixation, and the phosphate buffer was let stand for 45 minutes. 
     At step  5 E, dropping 25 μl of 0.1M phosphate buffer onto the WSB treated area cleans the treated area, and the phosphate buffer was let stand for 20 minutes. 
     At step  5 F, performing a series of dehydration stages on the WSB treated area by dropping 30% EtOH and letting stand for 15 minutes, then dropping 50% EtOH and letting stand for 15 minutes, then dropping 70% EtOH and letting stand for 15 minutes, then dropping 90% EtOH and letting stand for 15 minutes, then dropping 95% EtOH and letting stand for 15 minutes, and then dropping 100% EtOH and letting stand for 15 minutes, and then dropping on the WSB treated area once again 100% EtOH and letting stand for 15 minutes. 
     Under SEM, the analyte  32 , which in this experiment is bacteria, was found on a surface or the storage pockets of the ASW-15, ASW-25, and ASW-240. ASW-25 provided the best fixation images under SEM. No bacteria were found on the surface or storage packets of the ASW-50 and ASW-DC. In addition, “salt” particles were detected in some SEM images. Without being bound by the theory, the “salt” particles were possibly present on the SEM images because the fixation procedure was performed by dropping reagents onto the WSB rather than soaking the WSB. 
     PCR Analysis Using Water Soluble Biomaterial 
     A series of experiments were carried out to determine whether the WSB substantially inhibits with PCR reactions when the analyte  32  bound to the membrane  14  of the LFT device  10  is placed in the analysis system for PCR analysis. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. A Microfuge i.e., a “bench top centrifuge,” (Beckman Instruments, Inc.; Eppendorf, centrifuge 5418) was used. A PCR machine, namely PTC-200 Thermo Cycler (MJ Research, Inc.), was prepared. A puncher (Whatman® Harris Uni-Core™) was used to obtain discs for PCR amplification. 
     In Experiment 6, the objective is to determine whether the WSB substantially inhibits with PCR reactions. The exemplary method steps of a protocol for PCR analysis using analyte  32  fixed to the WSB is commenced. The exemplary analyte was a viral ds-RNA (double strand RNA), specifically CiLV-C (Citrus leprosis virus C) at 400 ng/μl concentration. Step  6 A includes spotting 1 μl of a viral RNA on the WSB is performed. 
     Step  6 B includes obtaining 1.2 mm disc of the viral spotted WSB. Step  6 C includes adding the 1.2 mm disc of each of the viral spotted WSBs to a separate PCR test tube containing RT-PCR reaction mix. The exemplary 20 μl of RT-PCR reaction mix:
         sterile nuclease-free water, 3.2 μl;   2× Reaction mix (Invitrogen), 10.0 μl;   RNAsin Plus 40 U/μl, 1.0 μl;   5 μM Forward Primer, 2.0 μl;   5 μM Reverse Primer, 2.0 μl;   SuperScript™ III One-Step RT-PCR System with Platinum® Tag DNA Polymerase (Invitrogen™ 12574-026) Mix, 0.8 μl;   and albumin from bovine serum (SIGMA-ALDRICH® A7888) at 10 μg/μl, 1.0 μl.       

     Thus, each test tube for the RT-PCR reaction contains 20 μl of RT-PCR mix and the 1.2 mm disc of the viral spotted WSB. 
     In Step  6 D, there is running a RT-PCR reaction under the conditions, as shown in the table 1 below: 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Step 
                 Temperature 
                 Time 
                 Number of Cycles 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 cDNA synthesis 
                 50° C. 
                 30 minutes 
                 1 
               
               
                 Initial denaturation 
                 94° C. 
                  2 minutes 
                 1 
               
               
                 Denaturation 
                 94° C. 
                 30 seconds 
                 40 
               
               
                 Annealing 
                 56° C. 
                 30 seconds 
               
               
                 Elongation 
                 72° C. 
                 30 seconds 
               
               
                 Final Elongation 
                 72° C. 
                  7 minutes 
                 1 
               
               
                   
               
            
           
         
       
     
     In Step  6 E, there is loading PCR products from each test tube from the reverse transcription PCR (RT-PCR) reaction in separate preparations of 1.0 to 2% Agarose gel in 1×TBE (Tris base, boric acid and EDTA) and running electrophoresis on the PCR products for 90 minutes at 90 Volts. Visualization was made by incorporating SYBr green to the agarose gel. The observation was made upon ultraviolet (UV) light. 
     Amplification by RT-PCR was successful using the viral spotted ds-RNA as the PCR template. In other words, the WSB does not substantially inhibit the PCR reaction. 
     In Experiment 7, the objective is to determine whether the WSB substantially inhibits PCR product quantification. ASW-25, ASW-50, and ASW-DC were tested. 
     The exemplary method steps of a protocol for PCR product quantification using analyte fixed to the WSB is commenced. Step  7 A is loading 2 μl of PCR product from Experiment 6 in the NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific of Wilmington, Del.), Nucleic Acid—DNA option for each PCR product produced from each WSB. Step  7 B is quantifying each PCR product produced from each WSB. The DNA quantification results were ASW-25, 379.7 ng/μl; ASW-50, 384.8 ng/μl; and ASW-DC, 365.9 ng/μl for. Recall that the viral ds-RNA tested was CiLV-C, 400 ng/μl. Thus, the WSB does not substantially inhibit the PCR reaction technique for quantification of PCR reaction product. 
     In Experiment 8, the objective is to determine what concentrations of DNA fixed to WSB can be amplified by PCR. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were tested. The template is  Pythium spinosum  (“PS-1”), 10 ng/μl. 
     The exemplary method steps of a protocol for serial dilution of DNA template and testing PCR amplification sensitivity is commenced. Step  8 A is transferring 10 μl to PS-1 to a test tube A. Step  8 B is transferring 1 μl from the test tube A to a test tube B that has 9 μl of H 2 O for 10 μl total volume in test tube B. Step  8 C is transferring 1 μl from the test tube B to a test tube C that has 9 μl of H 2 O for 10 μl total volume in test tube C. Step  8 D is transferring 1 μl from the test tube C to a test tube D that has 9 μl of H 2 O for 10 μl total volume in test tube D. Step  8 E is transferring 1 μl from the test tube D to a test tube E that has 9 μl of H 2 O for 10 μl total volume in test tube E. Step  8 F is transferring 10 μl of H 2 O to a test tube F. Steps  8 A to  8 F are repeated for each WSB tested. 
     At step  8 G, 1 μl from each of the test tubes  8 F is spotted on the WSB. Step  8 G is repeated for each WSB tested. 
     At step  8 H, a 1.2 mm disc of the spotted WSB area is taken by the puncher. Step  8 H is repeated for each WSB tested. 
     Step  8 I is adding each of the 1.2 mm discs from each of the spotted WSB to separate PCR test tube for each spotted WSB sample. The control has no WSB and 1 μl plasmid DNA for each concentration. The exemplary 20 μl of RT-PCR reaction mix: 
     sterile nuclease-free water, 8 μl; 
     GoTaq mix (Promega Corporation, Madison, Wis.), 10 μl; 
     10 μM Forward Primer, 1 μl; and 
     10 μM Reverse Primer, 1 μl. 
     In Step  8 J, there is running a RT-PCR reaction under the conditions, as shown in table X, for each separate PCR test tube for each spotted WSB sample. 
     In Step  8 K, there is loading PCR products from each test tube from the RT-PCR reaction in separate preparations of 1.0% Agarose gel and running electrophoresis on the PCR products for 90 minutes at 90 Volts. 
     As shown in Table 2 below, ASW-15, ASW-25, and ASW-50 did not interfere with PCR amplification up to the fourth dilution. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 Sample 
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 ASW- 
                 ASW- 
                 ASW- 
                 ASW- 
                 ASW- 
                 Positive 
               
               
                 Tube 
                 15 
                 25 
                 50 
                 DC 
                 240 
                 CONTROL 
               
               
                   
               
               
                 A 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 B 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 C 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 D 
                 + 
                 + 
                 + 
                 + 
                 − 
                 + 
               
               
                 E 
                 + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 F 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                 (negative 
               
               
                 control) 
               
               
                   
               
            
           
         
       
     
     The + signifies a positive result, i.e., PCR amplification successful. The − signifies a negative result, i.e., PCR amplification unsuccessful. The F test tube was a negative control for each of ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240, i.e., tube F is the negative control, it is required to demonstrate the WSB is not reactive or contaminated. 
     Exemplary Sample Pad Materials 
     After the format for the LFT device  10  was developed as shown in  FIG. 7 , a series of experiments were conducted to search for the best material for the sample pad  12 . The sample pad  12  and the wick  16  were made from the same material. The membrane  14  was made from ASW-25. The sample pads  12  were 26 mm long. The membranes  14  were 25 mm long. The wicks  16  were 21 mm long. The sample pads  12 , the membranes  14 , and the wicks  16  were 4 mm wide. The holder  46  was provided of length 8 mm on the backing card of 75 mm length and 4 mm width. See  FIG. 7  for length and width orientations. A test solution of 1×PBST 500 μl with 15 μl dye was used. 
     The objective of Experiment 9 was to determine which materials tested would produce fast flow through the LFT device  10 . The materials tested for the sample pad  12  included: Ahlstrom (Helsinki, Finland) 1660, 1662, 1663, 6615; Millipore (Billerica, Mass. USA) C048, C0083, G041; and Whatman (Whatman plc, UK) 16-S, 470, 2668, 2727, CF6, Fusion 5™ GF33, GB002, GF/D, Standard 17. ASW-15, ASW-25, ASW-50, ASW-DC, and ASW-240 were also tested for the sample pad  12 , although it was found that making the sample pad  12 , the membrane  14 , and the wick  16  from a single continuous piece was impractical, because the bottom part of the continuous piece that soaked in the test solution dissolved and mixed in the solution. Millipore C083; and Whatman GF33, GB002, and Standard 17 were good candidates for the sample pad  12  with wicking rate less than 4 minutes. In an exemplary embodiment, the less than 4 minute is preferable to reduce the possibility that the WSB in the membrane, in this case ASW-25, will dissolve in the solution during the wicking process. 
     In experiment 10, the four candidate materials for the sample pad  12  were tested again. Millipore C083 had the lowest wick rate and the smallest standard deviation when each of the good candidates were tested 10 times each; therefore, Millipore C083 was selected for the sample pad  12  in the LFT device  10  in one other exemplary embodiments. The Millipore C083 wicking rate mean is 2.9417 minutes. The Whatman Standard 17 wicking rate mean is 3.2266 minutes. The Whatman GB002 wicking rate mean is 3.7321 minutes. The Whatman GF33 wicking rate mean is 3.7928 minutes. 
     In other words, an exemplary embodiment includes a method comprising, the steps of: flowing a sample  26  solution that contains an analyte  32  through an LFT device  10  configured to bind the analyte  32 ; punching a sample disc  48  that binds the analyte  32  from the LFT device  10 ; and placing the sample disc  48  directly in an analysis system for analysis of the analyte  32 , in which the step of placing the sample disc  48  is not preceded by a step of eluting the analyte  32  from the sample disc  48 . A second embodiment includes the LFT device  10  includes at a least water soluble paper that binds the analyte  32 , and the sample disc  48  is punched from the water soluble paper. A third embodiment includes the analysis system includes at least an analysis technique selected from a group consisting of polymerase chain reaction (“PCR”), and the initial denaturation is a predetermined prolonged interval selected to promote the PCR. A fourth embodiment includes the step of flowing a sample  26  solution is preceded by at least the steps of: measuring a predetermined amount of a sample  26  material; calculating a volume of a solution responsive to the predetermined amount of the sample  26  material; providing a sample bag to which the volume of the solution is added; adding the predetermined amount of the sample  26  material to the solution in the sample bag; and grinding the sample  26  material in the bag until it is macerated and the solution is translucent to prepare the sample  26  solution. 
     While the invention has been described in connection with an exemplary embodiment, it is not intended to limit the scope of the invention to the particular form set forth, but on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. 
     It will be clear that the present invention is well adapted to attain the ends and advantages mentioned as well as those inherent therein. While presently preferred embodiments have been described for purposes of this disclosure, numerous changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed by the appended claims.