Abstract:
A fluidic device is disclosed for processing a biological sample in order to extract nucleic acids contained in said sample and for subsequently amplifying said extracted nucleic acids, said device including a processed sample storage archive area  10  comprising an absorbent solid substrate  14  treated with at least one nucleic acid stabilising reagent or reagent mix, said substrate allowing the generally dry and stabilised storage of said extracted and/or amplified nucleic acids, for example for long term storage of biological samples recovered forensically.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the storage of a sample processed within a fluidic device or the like. 
       BACKGROUND OF THE INVENTION 
       [0002]    WO2010091414 describes a microfluidic device which can biochemically amplify DNA from a biological sample by polymerase chain reaction (PCR) and store, in liquid form, a portion of the sample that is not used in the PCR, in what is called a ‘sample archive’. In that document it is proposed to seal the archive with paraffin wax for storage. 
         [0003]    The present inventor has recognised that the storage arrangement described in WO2010091414 has drawbacks, particularly, in terms of sample stability, and the likely need for low temperatures during extended storage, and the use of liquid seals which can leak. The present inventor has also realised that there is a need to provide more a robust storage device, which allows storage of a processed biological sample at room temperature for many years if necessary, but which is low cost and very reliable. Such an improved storage device would, for example, be ideal for storing processed samples collected forensically from a crime scene. 
       SUMMARY 
       [0004]    According to a first aspect, the present invention provides a fluidic device for processing a biological sample in order to extract nucleic acids contained in said sample and for subsequently amplifying said extracted nucleic acids, said device including a processed sample storage archive area comprising an absorbent solid substrate treated with at least one nucleic acid stabilising reagent or reagent mix, said substrate allowing the generally dry and stabilised storage of said extracted and/or amplified nucleic acids. 
         [0005]    Herein, a processed biological sample is one which has been subjected to some chemical or biochemical process, for example, where a sample is initially a raw crime scene sample, then processing will include initial purification and/or elution of a that sample. Processing will also include subsequent steps such as PCR steps. So the scope of this invention includes storage of nucleic acids exacted from biological samples for example via purification and/or elution processing steps but which have not yet undergone a PCR, as well as those nucleic acids which have been exacted and amplified, for example via a PCR. 
         [0006]    Herein, amplification of nucleic acids is not restricted to a PCR. Other known methods for multiplying or otherwise copying nucleic acids taken from biological samples are within the scope of this invention. 
         [0007]    According to a second aspect, the invention further provides a processed biological sample storage archive for generally dry storage of a nucleic acids extracted from a processed biological sample, the archive comprising a generally dry absorbent solid substrate treated with at least one nucleic acid stabilising reagent or reagent mix, said substrate allowing the generally dry and stabilised storage of said extracted and/or amplified nucleic acids, said archive being adapted for removable mounted to the fluidic device according to the first aspect. 
         [0008]    According to a third aspect, the invention provides a method of operating a biological sample processing fluidic device, in order to store a portion of the processed sample, the method comprising the following steps, in any suitable order: i) receiving a biological sample at a receiving chamber; ii) initially processing said sample, for example by purification and elution of nucleic acids in the sample; iii) directing a portion of said initially processed sample to a sample storage archive for generally dry storage of nucleic acids extracted from a biological sample, the archive comprising a generally dry absorbent solid substrate treated with at least one nucleic acid stabilising reagent or reagent mix; iv) optionally removing said storage archive and optionally removing the substrate from the remainder of the archive. 
         [0009]    The invention is further characterised by the claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]    The invention can be put into effect in numerous ways, illustrative embodiments of which are described below with reference to the drawings, wherein: 
           [0011]      FIG. 1  shows a sample archive according to the invention, mounted to a fluidic device; 
           [0012]      FIG. 2  shows another view of the sample archive shown in  FIG. 1 ; 
           [0013]      FIGS. 3 and 4  show top and bottom views respectively of the sample archive removed from the fluidic; 
           [0014]      FIGS. 5 a, b  and  c    show side views of the sample archive; and 
           [0015]      FIGS. 6 to 13  illustrate the method of use of the sample archive shown in the previous figures. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0016]    Referring to  FIG. 1 , a fluidic device  1 , for example principally as disclosed in WO2010091414 includes a removable plastics sample archive chamber  10  housed on the device  1 . The device  1  is shown transparently for clearer visibility of the archive  10 , although in practice it is likely not to be so. 
         [0017]    The archive chamber  10  sits inside the fluidic device  1 , in this case a DNA analysis chipset. Typically, extraction and purification involve the use of silica, with a chaotrope and organic solvent to elute the nucleic acids. Following sample extraction/purification of nucleic acids, a predetermined amount of that processed sample is delivered to an amplification chamber for a PCR, to amplifying any extracted nucleic acids. The process involves an enzymatic reaction using oligonucleotide sequences and subsequent electrophoresis. The remaining portion of the sample is delivered to the archive chamber  10  along a fluid path  12  in the direction of arrow B, where it is absorbed onto a solid fibrous storage medium  14 , in this case a treated cellulose paper-like matrix, for example as sold by Whatman Inc. under the trade name FTA®, housed releasably within the archive chamber  12 . The archive chamber is formed from a plastics frame  11 , which supports a flexible elastomeric seal  16  mounted to the periphery of the frame  11 . The seal  16  removably holds the archive  10  in place on the device and includes upper and lower lips  15 / 17  which provide a fluid-tight vacuum seal between the archive  10  and the device  1 . 
         [0018]    Referring additionally to  FIG. 2 , once the processing on the chipset is complete the archive chamber can be removed from the device  1 . In order to remove the archive  10  from the device  1  an implement can be inserted into a recess  19  in order to lever it out. The relatively small archive chamber  10  is easier to store and handle than the entire chipset which can then be disposed of. If required, the processed sample now stored on the substrate  14  can be removed from the archive chamber  10  and used in downstream processing using standard analysis techniques or it can be stored for future use, possibly many years later. The sample archive allows for generally dry storage and retrieval of a purified DNA sample extracted from a sample being processed on the device. 
         [0019]    In  FIG. 2 , an opening  13  of the fluid path  12  is visible which opens into the archive between the upper and lower lips and delivers the processed sample to the archive. 
         [0020]    Referring to  FIG. 3  the upper face of the now-removed archive  10  in shown. The archive  10  includes a transparent cover member  20  sealed to the frame  11  by a tamper-evident adhesive bond  22  around its periphery shown as a dotted line, so that it is evident that the cover member has, or has not been removed from the remaining archive. The adhesive bond  22  extends over the seal  16  also, so that it is possible to see whether or not the archive  10  has been removed from the device  1 . 
         [0021]    Referring to  FIG. 4 , the lower surface of the archive is shown. The archive includes an integrated RFID tag  24  that matches the identification of the microfluidic chipset  1 , for sample tracking purposes. The archive chamber  10  also includes a barcode  26  for the same purposes. Also included is an area  28  for labelling with a sticky label, marker pen or other indicia. 
         [0022]    Referring to  FIG. 5 a   , a side view of the archive  10  is shown schematically. In  FIG. 5 b   , the cover member  20  is shown partially removed, and in  FIG. 5   c,  the substrate  14  is shown removed in the direction of arrow X, from the remainder of the archive  10 . 
         [0023]      FIGS. 6 to 13  illustrate a method of processing a sample for use with the archive described above. 
         [0024]      FIG. 6 : according to the method, a DNA sample, for example from a buccal swab or a blood sample, is deposited into a receiving chamber  2  of a fluidic device  1 , in this case a microfluidic processing chipset, for processing. The sample is purified at a downstream location  3 , in this case by binding the DNA to a membrane, and carrying out multiple washing steps. The purified DNA is eluted from the membrane into a channel  8 . 
         [0025]      FIG. 7 : The processed sample in the form of eluted fluid F flows through the channel  8  in the direction of arrow A and then into a PCR chamber  4 . 
         [0026]      FIG. 8 : The PCR chamber  4  fills and becomes choked at its narrow exit  9 . 
         [0027]      FIG. 9 : The choking of the PCR chamber  4  prompts the flow of further processed sample fluid F to divert into the channel  12  in the direction of arrow B toward an open valve  5 . 
         [0028]      FIG. 10 : The fluid F flows past the open valve  5  and into the archive chamber  10  at the opening  13  where it is absorbed on the substrate  14 , in this case FTA paper as described above. 
         [0029]      FIG. 11 : The valve  5  is closed, to seal the archive  10 . 
         [0030]      FIG. 12 : The processed sample F is further processed by a PCR to amplify any nucleic acids in the sample fluid, for example according to known thermo-cycling and enzymatic techniques. 
         [0031]      FIG. 13  The further processed sample fluid F exits the PCR chamber  4  at exit  9 , for yet further processing, for example electrophoretic separation. The sample archive  10  can remain in the microfluidic device, or it can be removed as described above. 
         [0032]    Although embodiments have been described and illustrated above, it will be apparent to the skilled addressee that additions, omissions and modifications are possible to those embodiments without departing from the scope of the invention claimed. For example, although this invention can be implemented onto a microfluidic device which employs just a few millilitres of fluid, to allow recovery of a processed sample rather than sending the excess processed sample to waste, any fluidic device can utilise this invention. If used in a microfluidic device, this invention will be useful in forensics, in particular for crime scene samples, where the sampling opportunities may be limited, thereby preserving the sample. It will allow the same sample to be reinvestigated if a sample were to fail the initial analysis, but it would also give the option to perform other forensic analysis tests on the same sample such as mitochondrial DNA, YSTR, SNP analysis. The fact that the sample is initially processed, for example purified, will save time and cost of performing these extra tests. 
         [0033]    One specific example of the substrate  14  has been given as FTA®, which has been chosen because its treatment inhibits the degradation of DNA. In one embodiment, the substrate treatment is wet-applied stabilising reagents in the form of combination of a weak base, and a chelating agent, optionally, uric acid or a urate salt, and optionally an anionic surfactant. 
         [0034]    It is preferred that the weak base is a Lewis base which has a pH of about 6 to 10, preferably about pH 8 to 9.5. 
         [0035]    Alternatively, the weak base is an organic and inorganic base, and if inorganic then optionally includes an alkali metal carbonate, bicarbonate, phosphate or borate (e.g. sodium, lithium, or potassium carbonate), and if organic then optionally includes, tris-hydroxymethyl amino methane (Tris), ethanolamine, tri-ethanolamine and glycine and alkaline salts of organic acids (e.g. trisodium citrate). 
         [0036]    Alternatively, the weak base is Tris present either as a free base or as a salt, for example, a carbonate salt. 
         [0037]    Preferably, the chelating agent binds multivalent metal ions with a comparable or better affinity than ethylene diamine tetraacetic acid (EDTA), and is preferably EDTA. 
         [0038]    Preferably, the anionic surfactant includes a hydrocarbon moiety, aliphatic or aromatic, containing one or more anionic groups. 
         [0039]    Preferably, the anionic surfactant is a detergent, for example sodium dodecyl sulphate (SDS) and/or sodium lauryl sarcosinate (SLS). 
         [0040]    Other stabilising reagents could be employed, for example a chaotropic substance such as a chaotropic salt, for example guanidinium thiocyanate. 
         [0041]    The substrate  14  is treated with the stabilising reagents mentioned above so as to be capable of carrying out several functions: (i) lyse intact cellular material upon contact, releasing genetic material, (ii) enable and allow for the conditions that facilitate genetic material immobilization to the solid support (probably by a combination of mechanical and chaotrophic), (iii) maintain the immobilized genetic material in a stable state without damage due to degradation, endonuclease activity, UV interference, and microbial attack, and (iv) maintain the genetic material as a support-bound molecule that is not removed from the solid support during any downstream processing. It will be apparent to the skilled addressee that other reagent mixes could perform one or more of the functions mentioned above. 
         [0042]    It is possible that the processed sample could be stored in the archive after PCR amplification, where an amplified or otherwise copied DNA sequence is required.