Sampling core

Abstract

A method for producing a sampling device for analyzing a biological sample is described, the method comprising adsorbing and drying a liquid reaction mixture into an elongated porous matrix, and segmenting the porous matrix into regular segments.

Description

Technical Field

[0001] The present invention relates to a method for producing a sampling device for analyzing a biological sample. Background Art

[0002] Nucleic acid amplification diagnostic tests are commonly used around the world, especially since the SARS-CoV-2 pandemic has led to the development of polymerase chain reaction (PCR) testing as the gold standard for diagnosing infection. However, PCR testing has many disadvantages and other diagnostic techniques are being explored, including isothermal amplification methods, which have the advantage of not requiring thermal cycling and can be performed at room temperature in some cases.

[0003] Such isothermal amplification methods require the provision of suitable reagents, such as enzymes and reaction mixtures, and this may also pose problems for the storage and supply of the reagents. If provided in wet form, temperature-controlled storage may be required, while if provided in dry form, reconstitution with accurately measured volumes of water may be required.

[0004] Furthermore, implementation of a classical isothermal amplification assay may also require accurate and precise measurement of sample volumes.

[0005] One approach that has been used for such diagnostic assays is to provide lyophilized reagents on an absorbent matrix; a wet sample (e.g., blood or saliva) can be delivered to the matrix, which will rehydrate and reconstitute the lyophilized reagents. However, this may also require accurate measurement of sample volumes to ensure that the reagents are reconstituted at the correct concentration.

[0006] It would be beneficial to provide alternative materials for use in diagnostic assays, particularly (but not exclusively) for isothermal amplification of nucleic acids; and to provide methods of making such materials. Summary of the invention

[0007] The present invention at least partially satisfies these needs by providing a method for producing a sampling device for analyzing a biological sample, the device being formed of a porous matrix and comprising dried reagents for performing the analysis. The inventors unexpectedly determined that such a method can provide dried reagents that are fully compatible with downstream assays and analyses, including isothermal nucleic acid amplification assays.

[0008] In a first aspect, there is provided a method for producing a sampling device for analyzing a biological sample, the method comprising:

[0009] a) providing a porous matrix having a regular cross-section and generally an elongated form;

[0010] b) contacting the substrate with a liquid reaction mixture such that the reaction mixture is adsorbed into the substrate;

[0011] c) drying the adsorbed liquid reaction mixture to provide a porous matrix having the dried reaction mixture therein; and

[0012] d) dividing the porous matrix into one or more sections of predetermined length;

[0013] The sampling device is thus provided.

[0014] In use, the sampling device may contact with a biological sample, which is a liquid sample (e.g., blood, saliva, serum, sputum, urine) or a dry sample that has been rehydrated with a suitable liquid (e.g., water, reaction buffer, etc.). Due to the porous nature of the matrix, the sample will be adsorbed into the matrix, and the dried reaction mixture is rehydrated to allow for related determinations (e.g., isothermal amplification) to be performed in the matrix. Although the device can be introduced into the sample classically, such as by dipping the device into or immersing it in a liquid sample, in some embodiments, the sample can be introduced into the device (e.g., by placing or distributing the sample previously obtained on the device). In addition, the device can be left in place for subsequent reaction and determination, or can be transported to a separate location (e.g., loaded into a determination reading device) for subsequent reaction and determination.

[0015] The step of segmenting the porous substrate into segments may include cutting the porous substrate into separate segments. However, in some embodiments, these segments may remain physically connected after segmentation, but are not fluidically connected. In particular, when the substrate is a polymeric material, the segmentation step may take the form of curling the continuous substrate into segments; optionally, heating is performed simultaneously to melt the polymer in the curled zone. This method can be performed at a sufficiently short time and a sufficiently low temperature to avoid denaturing the associated enzymes in the substrate.

[0016] As can be seen from the description herein, this method of manufacture has many advantages and benefits. A key advantage is that this method allows for continuous manufacture - the elongated matrix can be sequentially passed through a reaction mixture and drying station to a cutting station where segments can be cut in step d); this can provide multiple sampling devices in a relatively short time.

[0017] Another advantage comes from cutting or otherwise dividing segments of the porous matrix into different lengths. Since the matrix has a regular cross-section, the volume of the cut segment per unit length will be defined; this, combined with known properties such as the void volume and air space of the matrix, means that each cut segment will absorb a consistent volume of liquid. This in turn produces the key advantage that the volume of the reaction mixture absorbed into the matrix and dried will match the volume of the sample absorbed by the same segment; allowing the concentration of the reaction mixture to be controlled to achieve the optimal dilution of the sample for the desired reaction.

[0018] The matrix is ​​preferably a fibrous matrix comprising a plurality of bonded fibers defining voids therein. The fibrous matrix may be an extruded fibrous matrix. The fibers in the matrix are preferably aligned with each other to define a longitudinal axis, which is typically the same axis as the long axis of the elongated matrix. The fibers may be a polymer material, and are preferably polyolefins (including LDPE, LLDPE, HDPE, PP); polyesters (including PET, PTT, PBT); or polyamides. The most preferred polymer is polyethylene or polypropylene. In an embodiment, the pore size of the matrix is ​​between 10-100 μm and the pore volume is 50-95%. Suitable materials include materials manufactured by Porex Corporation, Georgia, USA.

[0019] A particularly preferred matrix is ​​described in US2005 / 0189292, the contents of which are incorporated herein by reference. Specifically, this matrix comprises a self-sustaining fluid transport body comprising a plurality of bundled, curled bicomponent fibers bonded to each other at spaced apart contact points, each bicomponent fiber having a fiber structure comprising a first fiber component and a second fiber component formed of a polyamide material, the fibers together defining a tortuous fluid flow path through the fluid transport body. Another benefit of using an extruded fiber matrix is ​​that the resulting matrix generally provides a "core" and "sheath" arrangement, wherein the matrix is ​​generally denser around the circumference and less dense in the center; this helps to reduce the loss of sample fluid due to the circumferential edge of the cutting device and promotes the flow of sample fluid from the cutting surface into and along the device; and reduces the loss of dry reagent mixture during handling and transportation. The preferred composition of this matrix for use in the present invention is a polyethylene core and a polypropylene sheath. The use of polypropylene is particularly advantageous because polypropylene does not bind proteins; this means that reagents can be dried onto the matrix and rehydrated without the risk of significant loss due to binding to the matrix. In some embodiments, the reagent may additionally comprise bovine serum albumin (BSA), which has been shown to further reduce non-specific protein binding to the matrix.

[0020] Natural fibers with the potential to reduce environmental impact, such as cellulose and lignin, may also be considered. See, for example, the materials discussed in "A review on natural fibers for development of eco-friendly bio-composite: characteristics, and utilizations", Karimah et al., Journal of Materials Research and Technology, Vol. 13, July-August 2021, pp. 2442-2458.

[0021] The reaction mixture can be used for any suitable assay, but is preferably used for an assay based on nucleic acid amplification. This can include an isothermal nucleic acid amplification-based assay, including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), damage-induced DNA amplification (LIDA) and RT versions of these. The reaction mixture typically includes appropriate nucleotides, enzymes, primers and probes, as well as buffers, cations, etc. The skilled person will understand which components are necessary for a given assay.

[0022] In some embodiments, the reaction mixture is used for thermal cycling assays, such as conventional PCR methods, including but not limited to PCR, RT-PCR, LATE-PCR, and convection PCR (cPCR) and capillary loop convection PCR (clcPCR). The concept of convection has been used for DNA amplification based on PCR. A high surface area ratio cylinder and two heating zones set to denaturation (95°C) and annealing (50-70°C) cause amplification movement through the Rayleigh-Bénard convection phenomenon, thereby generating a stable flow and circulation in the temperature field. Therefore, there is no need to repeatedly heat and cool the container, and the reagents circulate spontaneously and then undergo three amplification processes. This technology is further modified to allow capillary loop convection polymerase chain reaction (clcPCR) to improve amplification efficiency. It is believed that efficiency is improved by generating directional flow. This uses a U-shaped ring glass capillary with an inner diameter of 1.6 mm. The clcPCR platform utilizes an isothermal heater to heat the bottom of the loop capillary.

[0023] In embodiments, the reaction mixture further comprises a cryoprotectant sugar to protect the protein from drying. Typical sugars for this purpose include disaccharides, such as maltose, sucrose, and preferably trehalose. Alternatively, the cryoprotectant sugar can be a high molecular weight sugar, such as dextran. In some embodiments, there may be a combination of sugar and / or other cryoprotectants, such as trehalose and dextran.

[0024] In embodiments, preferably the concentration of the reaction mixture before drying is a 1x concentration. This means that after rehydration with a predetermined volume of liquid sample absorbed by the device, the reconstituted reaction mixture is at the correct concentration to perform the assay.

[0025] Preferably, the reaction mixture comprises a fluorophore or chromophore to allow visual detection of the product of the reaction.

[0026] The step of contacting the fibrous substrate with the liquid reaction mixture may comprise immersing a portion of the substrate in the reaction mixture. Alternatively, the step may comprise spraying the portion of the substrate with the reaction mixture. In either case, the method may comprise allowing the immersed or sprayed portion to become saturated with the liquid mixture; this again maintains consistency between different devices.

[0027] The method may further include the step of covering the circumferential edge of the matrix with an impermeable coating. This may be performed before or after step d) cutting the matrix. The impermeable coating may be used to reduce the loss of sample fluid due to the circumferential edge of the cutting device, and promote the sample fluid to flow from the cutting surface and flow along the device; and reduce the loss of dry reagent mixture during handling and transportation. The coating may be, for example, a polymer wrap, a spray, etc.; or an extruded polymer tube; etc. Alternatively, as described above, the matrix may be manufactured in such a manner: having a denser sheath formed around the edge of a less dense core.

[0028] In some embodiments, the method can further include the step of assembling multiple sampling devices so that they are in fluid contact. This allows fluid to flow from the first sampling device to the second sampling device, and can allow different reaction mixtures to be included in each device. For example, the first device may be ready to include a reaction mixture for pre-treating a sample; for example, removing contaminants and / or lysing cells to release nucleic acids. Then, the second device in fluid contact with the first device can receive the pre-treated sample and perform, for example, a nucleic acid amplification reaction. In some embodiments, another device can include a reagent for detecting a reaction product (e.g., amplified nucleic acid). The assembly step can include enclosing multiple devices in a laminated structure.

[0029] In some embodiments, the method can further include the step of assembling multiple devices so that they are in physical contact but not fluid contact. This allows multiple samples to be processed in parallel to increase throughput. For example, multiple cutting devices can be combined in the same direction to expose the cutting surface receiving the sample. The sample can then be applied to all devices simultaneously. The assembly step can include enclosing multiple devices in a laminated structure.

[0030] Another aspect of the invention provides a sampling device for analyzing a biological sample, the device comprising a segment of a porous matrix having a regular cross-section and generally an elongated form, the matrix containing a dried reaction mixture comprising reagents for analyzing the biological sample.

[0031] A segment may be physically and fluidically separated from other such segments; or may be fluidically separated but physically connected. In some embodiments, the device comprises a plurality of fluidically separated segments. In other embodiments, the device comprises a plurality of fluidically connected segments. Each of the segments may contain the same or different dried reaction mixture therein.

[0032] The matrix is ​​preferably a fibrous matrix comprising a plurality of bonded fibers defining voids therein. The fibrous matrix may be an extruded fibrous matrix. The fibers in the matrix are preferably aligned with each other to define a longitudinal axis, which is typically the same axis as the long axis of the elongated matrix. The fibers may be a polymer material, and are preferably polyolefins (including LDPE, LLDPE, HDPE, PP); polyesters (including PET, PTT, PBT); or polyamides. The most preferred polymer is polyethylene or polypropylene. In an embodiment, the pore size of the matrix is ​​between 10-100 μm and the pore volume is 50-95%. Suitable materials include materials manufactured by Porex Corporation, Georgia, USA.

[0033] Particularly preferred matrices are described in US2005 / 0189292, the contents of which are incorporated herein by reference. Specifically, this matrix comprises a self-sustaining fluid transport body comprising a plurality of bundled, curled bicomponent fibers bonded to each other at spaced apart contact points, each bicomponent fiber having a fiber structure comprising a first fiber component and a second fiber component formed of a polyamide material, the fibers together defining a tortuous fluid flow path through the fluid transport body. A preferred composition for this matrix of the present invention is a polyethylene core and a polypropylene sheath. In some embodiments, the agent may additionally comprise bovine serum albumin (BSA).

[0034] The reaction mixture can be used for any suitable assay, but is preferably used for an assay based on nucleic acid amplification. This can include an isothermal nucleic acid amplification-based assay, including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), damage-induced DNA amplification (LIDA) and RT versions of these. The reaction mixture typically includes appropriate nucleotides, enzymes, primers and probes, as well as buffers, cations, etc. The skilled person will understand which components are necessary for a given assay.

[0035] In some embodiments, the reaction mixture is used in a thermal cycling assay, such as conventional PCR methods, including but not limited to PCR, RT-PCR, LATE-PCR, as well as convection PCR (cPCR) and capillary loop convection PCR (clcPCR).

[0036] In embodiments, the reaction mixture further comprises a cryoprotectant sugar to protect the protein from drying. Typical sugars for this purpose include disaccharides, such as maltose, sucrose, and preferably trehalose. Dextran may also or alternatively be present.

[0037] In embodiments, preferably the concentration of the reaction mixture prior to drying is a 1x concentration.

[0038] Preferably, the reaction mixture comprises a fluorophore or chromophore to allow visual detection of the product of the reaction.

[0039] Upon contact with the liquid sample, the sample is drawn into the device by capillary action to a predetermined volume required to rehydrate the reaction mixture to a concentration suitable for conducting the reaction.

[0040] In embodiments, the porous matrix acts as a light guide to transmit and direct the visual signal from the reaction; inclusion of a sugar, preferably trehalose, that matches the refractive index of the porous matrix may also improve the light guiding properties.

[0041] In some embodiments, the porous matrix comprises one or more crowding agents or cosolvents for simulating macromolecular environments to improve local reaction kinetics. That is, the crowding agent or mimetic, possibly together with the geometry of the matrix, can ensure that the reagents are clustered together and the reaction can proceed in an accelerated manner compared to the reaction in the absence of the reagent or mimetic. It is believed that one such crowding agent mimetic is trehalose.

[0042] Another aspect of the invention provides a method of enhancing reaction kinetics in a diagnostic reaction, the method comprising conducting the diagnostic reaction within a porous matrix in the presence of a crowding agent or co-solvent for simulating a macromolecular crowded environment, such that the reaction is accelerated compared to the same diagnostic reaction conducted in a liquid in the absence of the crowding agent or crowding agent mimetic. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] These and other aspects of the present invention will now be described with reference to the accompanying drawings, in which:

[0045] Figure 1 An illustration showing the production process of the device

[0046] Figure 2 Illustrate the cutting and curling of porous substrates

[0047] Figure 3 Shows the use of a crimping device with separate zones for different reactions

[0048] Figure 4 Arrangement showing separation of lysis and detection

[0049] Figure 5 Multiple stacked devices shown

[0050] Figure 6 Shows the use of the device in the bracket

[0051] Figure 7 Further views illustrating the device in the bracket

[0052] Figure 8 Showing the effect of incorporating trehalose in a test RT-LAMP amplification reaction

[0053] Fig. 9 Demonstrated use of the wick as a light guide to direct the fluorescent signal

[0054] Detailed Description of the Drawings

[0055] Lyophilization (freeze drying) is a low temperature dehydration process that involves freezing the product and using a vacuum to remove the ice by sublimation; while it can be used to produce dry reagents, it can be complicated to implement. While developing sampling cores for conducting bioassays, the inventors determined that a more direct dehydration method using heat to evaporate water was feasible and produced sampling cores containing dry reagents that could be used in nucleic acid amplification and detection methods. This simple method provides access to a very simple continuous in-line manufacturing process, such as Figure 1 exemplified.

[0056] As shown, the method starts with a continuous roll of porous substrate - in this case, the porous substrate is 3 mm in diameter. An extruded fibrous polypropylene matrix begins. It is unrolled and passed through a spray station which saturates the segments of the core passing through the spray with a 1x concentration of liquid reagent solution. The specific reagent solution chosen will depend on the assay to be performed. After saturation, the core passes through a drying station which gently heats the core to evaporate the liquid, leaving behind a dry reagent. Thereafter, the core is either cut into individual pieces or a heat crimp is used to separate the continuous core into multiple connected segments. See also Figure 2 .

[0057] Although relatively simple, this method provides a variety of surprising advantages for the subsequent use of the core in sampling and analysis. The method provides a continuous true process of drying of the ultimate volume. Further, the high volume to surface area ratio allows the drying of 1x reaction mixtures, rather than higher concentrations of raw materials that are usually dried or freeze-dried due to thermal stress; therefore, this can avoid the common kinetic compromises when using high concentration buffers (e.g., 10x) due to the rehydration and mixing requirements that may be slow in capillary formats.

[0058] This method also eliminates the need for accurate pipetting on the production line. This advantage is particularly important when pipetting concentrated stocks, where volume errors are important to maintain the final concentration. In addition, highly concentrated enzyme stocks are no longer required, reducing issues associated with glycerol, which can be problematic.

[0059] However, a key advantage is that the reagent core can be cut to length and become a discrete and self-regulating component, which also eliminates the need for accurate pipetting of samples. Instead, the sample is drawn into the core by capillary action to the exact volume required to fill the core and rehydrate the reagent, because the core that has absorbed the correct volume of reaction buffer can only draw a set volume defined by its geometry. Therefore, the core becomes its own predictive sample and reagent container. Since the dried reagent is within the fiber matrix, there is no problem of reagent knocking out during transportation.

[0060] To use the core, it can be contacted with a liquid sample. Adding the sample rehydrates the reagent in the core, and (as with reagent filling) the need for accurate pipetting is completely eliminated because the core only absorbs a limited volume defined by its geometry. Once the core is saturated with buffer (or water or sample), the excess is not absorbed by allowing excess sample to overflow; this can be used to accommodate changes in buffer volume delivery. In addition, since the core is filled from one or both ends to become saturated, it does not need to be oriented or have a directional filling in order to perform the assay. In some embodiments, the core can be immersed in or in contact with the sample; in other embodiments, the sample can be placed on or adjacent to the core to allow absorption.

[0061] One consideration when dehydrating or lyophilizing reagents is that protective agents may need to be included to prevent or reduce thermal enzyme denaturation. Sugar excipients are commonly used in both methods to protect proteins such as enzymes; in particular, trehalose is used by yeast and insects to provide thermal protection by replacing water molecules around proteins and prevent protein damage from extreme temperatures. Trehalose has been widely used in industry in combination with other sugars to provide room temperature stable enzymes for diagnostics. Other disaccharides have previously been shown to have similar properties. To ensure that the inclusion of such sugars did not affect the proposed nucleic acid amplification and diagnostic assays, sample tests were run. Figure 8 The effect of different concentrations of trehalose on the amplification performance of the tested RT-LAMP is shown. Figure 8It was shown that trehalose did not inhibit RT-LAMP even at high concentrations (300 mM, 10% v / v), and at this concentration 10 PFU / mL of the test sample could be detected within 1200 seconds. We believe that the use of concentrations of 70 mM, 150 mM, 300 mM, and 500 mM (15% v / v) should be feasible, and that trehalose alone or in combination with one or more other sugars may be included here. In addition to disaccharides, high molecular weight sugars such as dextran are particularly useful.

[0062] Additional data (not shown) using a pH color indicator within the core (ie, incorporated into the reaction mixture) to confirm that amplification had occurred, showed that after 20 minutes of RT-LAMP for the test sample at 10 PFU / mL, a visual indication of color change in the core was clearly visible.

[0063] Another potential advantage comes from the unexpected observation that the fibers in the matrix (particularly polyethylene fibers) can be used as light guides to transmit and direct fluorescence from the amplification and post-detection signal. When tested, surprisingly, there was very good optical coupling to the core fiber matrix, which enabled good fluorescence measurements by the optical reader. Without wishing to be bound by theory, it is believed that the high trehalose / sugar content may help with the refractive index matching of the filter fiber, allowing the photometer to interrogate / measure light from a larger proportion or from the entire reaction mixture.

[0064] See also Fig. 9 , which compares fluorescence in wet and dry cores, showing that light is transmitted to the end. Incorporation of additional sugars could potentially be used to tune the refractive index and further improve transmittance, if desired.

[0065] This conclusion suggests potential configurations for cores contained within plastic supports or containers. See, for example, Figure 6 .exist Figure 6 In the present invention, a single or multiple cores, each with a different fluorophore in the reaction mixture, are placed in adjacent wells in a plastic holder. When sample is added to saturate the cores, the residual sample liquid can accumulate to the sides of a single core, or can accumulate on top of multiple cores if the cores fill the sample holder. When the amplification reaction is carried out, the fluorescent signal will be transmitted along the core to exit from the cut surface; the fluorescent signal can then be detected using a photometer or other optical reader. This arrangement allows multiple reactions to be separated into separate different reactions by physical separation, and allows a single optical detector to query multiple reactions. Moreover, the core absorbs predetermined volumes of sample and reagents, eliminating the need for accurate liquid handling by the user.

[0066] Another advantage of placing the core in a plastic holder is that, since the sample is absorbed into the core by capillary action, the orientation of the holder is less important, and rotation or inversion of the holder and core will not affect the results. Figure 7 .

[0067] Furthermore, the diffusion rate along the length of the core is slow compared to the reaction rate. Therefore, excess sample can be located above the core without interfering with the reaction within the core.

[0068] Additional testing and development revealed the reaction kinetics advantages of the core format. Because the reagents are thinly coated on a single fiber and quickly and evenly resuspended, the distribution of the reagents in the sample is inherently uniform upon rehydration, and no mixing or stirring is required. The capillary structure can provide a microenvironment with improved kinetics or efficiency, involving local crowding agents (such as trehalose, etc.), and the short distance between fibers ensures a short diffusion distance to the target molecule, speeding up the reaction time and also ensuring comprehensive contact with the sample. Tests showed that the core provided a strong fluorescent signal, and the positive time (TTP) of the dried core was significantly improved (300 seconds) compared to conventional wet tube reactions. This was confirmed by RPA (recombinase polymerase amplification) and RT-LAMP. High concentrations of sugar-based cosolvents (such as dextran, trehalose, sucrose, sorbitol, and glycerol) can be used to simulate macromolecular crowded environments.

[0069] The construction and production methods of the cores described herein also lend themselves to the generation of cores with discrete "zones" for different parts of the amplification / detection method. For example, Figure 4 A single core is shown with a lysis reagent incorporated at a first end and a detection reagent incorporated at a second end, wherein the core is cut to the appropriate size to contain the two zones. Although the manufacturing method is more complicated (given the need to introduce two different reaction mixtures), the end use of the core may be simplified because the sample can flow through the core from the lysis end to the detection end. In a variation, lysis can occur in the central portion and the first sampling end can be untreated so that it is inert to the sample.

[0070] This works because the rate of diffusion from end to end through the capillary structure is much slower than the reaction kinetics of the events occurring in each zone.

[0071] Alternatively, if Figure 5 As shown, multiple individual cores, each containing a different reactive chemistry, can be stacked and connected together to allow a single sample to be processed sequentially in a variety of different ways as it passes through multiple cores.

[0072] Related arrangements such as Figure 3As shown; here, instead of multiple separate cores, two coiled sections remain connected, with the first section dedicated to lysis and the second section for detection. If the reagents are dried into the core after coiling (rather than before), the coil will prevent the reagents from flowing through during the manufacturing process, potentially simplifying the process of preparing multiple zones. The sample can be pushed into or pulled out of the core - for example, by placing the core into the tube of a pipette - so that the liquid bypasses the coil by leaving and re-entering the core.

Claims

1. A method for producing a sampling device for analyzing a biological sample, the method comprising: a) providing a porous matrix having a regular cross-section and generally an elongated form; b) contacting the substrate with a liquid reaction mixture such that the reaction mixture is adsorbed into the substrate; c) drying the adsorbed liquid reaction mixture to provide a porous matrix having the dried reaction mixture therein; and d) dividing the porous matrix into one or more segments of predetermined length; The sampling device is thus provided.

2. The method of claim 1, wherein the step of dividing the porous matrix into segments comprises cutting the porous matrix into individual segments.

3. The method of claim 1, wherein the segments of the porous matrix remain physically connected but not fluidly connected after segmentation.

4. A method according to any one of the preceding claims, wherein the porous matrix is ​​a fibrous matrix comprising a plurality of binder fibers defining voids therebetween.

5. The method of claim 4, wherein the fibrous matrix is ​​an extruded fibrous matrix.

6. A method according to claim 4 or 5, wherein the fibers in the matrix are polymeric materials, preferably polyolefins (including LDPE, LLDPE, HDPE, PP); polyesters (including PET, PTT, PBT); or polyamides.

7. A method according to claim 4, 5 or 6, wherein the fibres in the matrix are polyethylene and / or polypropylene.

8. A method according to any one of claims 4 to 7, wherein the fiber matrix comprises a polyethylene core and a polypropylene sheath.

9. The method according to any one of the preceding claims, wherein the reaction mixture is suitable for performing a nucleic acid amplification based assay.

10. A method according to any one of the preceding claims, wherein the reaction mixture comprises a cryoprotectant sugar to protect the protein from drying out, preferably trehalose.

11. The method according to any one of the preceding claims, wherein the concentration of the reaction mixture before drying is 1x concentration.

12. The method according to any one of the preceding claims, wherein the reaction mixture comprises a fluorophore or a chromophore to allow visual detection of the product of the reaction.

13. A method according to any one of the preceding claims, wherein the step of contacting the porous substrate with the liquid reaction mixture comprises immersing a portion of the substrate in the reaction mixture.

14. The method of any one of claims 1 to 12, wherein the step of contacting the porous substrate with the liquid reaction mixture comprises spraying portions of the substrate with the reaction mixture.

15. A method according to any one of the preceding claims, wherein the method comprises allowing the porous matrix to become saturated with the liquid mixture before drying.

16. A method according to any preceding claim, further comprising the step of assembling a plurality of sampling devices such that they are in fluid contact.

17. A method according to any one of the preceding claims, further comprising the step of assembling a plurality of devices such that they are in physical contact but not fluid contact.

18. A sampling device for analysing a biological sample, the device comprising a segment of a porous matrix of regular cross-section and generally elongated form, the matrix containing a dried reaction mixture comprising reagents for analysing a biological sample.

19. The sampling device of claim 18 comprising a plurality of fluidly separated said sections.

20. The sampling device of claim 18, comprising a plurality of fluidly connected said segments.

21. The sampling device of claim 19 or 20, wherein each of the segments contains a different dried reaction mixture therein.

22. The sampling device according to any one of claims 18 to 21, wherein the matrix is ​​a fibrous matrix comprising a plurality of binder fibers defining interstices therebetween.

23. The sampling device according to any one of claims 18-22, wherein the reaction mixture is suitable for a nucleic acid amplification based assay.

24. The sampling device according to any one of claims 18 to 23, wherein the reaction mixture further comprises a cryoprotectant sugar to protect the protein from drying, preferably trehalose.

25. The sampling device of any one of claims 18-24, wherein the reaction mixture comprises a fluorophore or a chromophore to allow visual detection of the product of the reaction.

26. The sampling device according to any one of claims 18 to 25, wherein: Upon contact with the liquid sample, the sample is drawn into the device by capillary action to a predetermined volume required to rehydrate the reaction mixture to a concentration suitable for conducting the reaction.

27. A sampling device according to any one of claims 18 to 26, wherein the porous matrix acts as a light guide to transmit and direct visual signals from the reaction.

28. A sampling device according to claim 27, wherein the device comprises a sugar, preferably trehalose, matching the refractive index of the porous matrix.

29. The sampling device of any one of claims 18-28, wherein the porous matrix comprises one or more crowding agents to improve local reaction kinetics.

30. The sampling device of any one of claims 18-29, wherein the device comprises a plurality of reaction zones, each reaction zone comprising a different dried reaction mixture therein.

31. A method of enhancing reaction kinetics in a diagnostic reaction, the method comprising conducting the diagnostic reaction within a porous matrix in the presence of a crowding agent or co-solvent for simulating a macromolecular crowded environment such that the reaction is accelerated compared to the same diagnostic reaction when conducted in a liquid in the absence of the crowding agent or crowding agent mimetic.

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