Patent Application
20080124720
This invention relates to a method for processing and analysing samples and an apparatus for carrying out the method. In particular, the invention relates to a combination of sequential processing steps self-contained within a housing. The invention can be used in relation to any sample analysis where processing of the sample with one or more reagents is required. The invention has particular relevance for DNA analysis.
In many fields of technology, samples of materials must be removed from a source and analysed, often following treatment with one or more chemical reagents or exposure to specific physical conditions. For example, there is a great need for the DNA analysis of biological samples in a wide variety of technology fields and industries.
The sample must first be procured from its source, the sample chemically and physically treated to prepare the DNA contained in the sample, the DNA then mixed with reagents to commence DNA-specific reactions (such as standard PCR methodology), and the products of such reactions analysed by physical or chemical methods. There are many known laboratory methods and devices available for achieving the individual steps, but none capable of performing the necessary and complex processes within a single device outside of the laboratory environment.
Further, it is often necessary to analyse a large number of samples, for example in food processing production lines. It is therefore desirable to use a sampling and analysis method that can be automated at least in part.
One problem associated with some sample processing and analysis techniques is the time required to obtain the results of analysis. For example, standard PCR analysis of a biological sample typically takes 2 to 6 hours. To meet the demands of systems and processes used in many industries, there is a need for a sampling and analysis method that is rapid.
The stringent requirements of health and safety, particularly in the human health and food industries, mean that cross-contamination between samples must not occur. It is therefore desirable to use a sampling and analysis apparatus that is not reusable and is disposable.
Further, it is desirable to analyse samples without high levels of technical skill and training required by operators. A fully self-contained apparatus having equipment and materials embedded within obviates the need for the high skill and experience levels needed by operators of known sampling and processing devices. A fully self-contained apparatus also has the advantage of being able to be stored for extended periods.
It is therefore an object of the invention to provide a method of sample analysis that overcomes at least in part any of the above mentioned problems or disadvantages, or to at least provide a useful alternative.
In one aspect of the invention there is provided a method for analysing a sample including:
The sample may be any sample of biological or non-biological chemical substances where processing with other reagents is needed prior to detection or measurement.
In a preferred embodiment of the invention the sample contains DNA. The invention is particularly suited to PCR analysis of the DNA. Typical examples of a sample include manufactured DNA samples and samples of blood, serum, saliva, urine, and milk, and an extract obtained from bone, faeces, fat, flesh, hair, skin, plant material, microbes, or microbial habitats.
The at least one sample processing reagent may be an encapsulated aqueous solution of one or more chemical or biochemical reagents required for DNA analysis including oligonucleotides, deoxynucleoside triphosphates and thermostable DNA polymerase.
Preferably the solid or semi-solid material is a wax or grease, for example paraffin, that is phase separated from any aqueous solution of sample, reagents, or products.
The detection step may use any suitable means for detecting or measuring the concentration of the product, but preferably uses an optoelectronic means.
Preferably the optoelectronic means is a light path between the sample and an optical detector which is connected to a microprocessor.
The at least one sample processing reagent is preferably caused to move along the conduit by the application of increased or reduced pressure to the conduit.
In a preferred embodiment of the invention a biological sample containing DNA is analysed according to the following steps:
Preferably the above steps 7 to 10 are repeated one or more times, typically 20-40 times.
In a second aspect of the invention there is provided an apparatus for analysing a sample including:
In a preferred embodiment of the invention, the sampling device includes a free rolling ball held within a socket where part of the external surface of the ball is capable of contact with a surface to obtain a sample from that surface by rolling the ball across the surface.
Preferably the shaft of the sampling device is a tapered longitudinal stylus, having a sample inlet in open communication with the socket and a conduit extending the length of the stylus.
The invention further provides a sampling device adapted for use in the apparatus of the second aspect of this invention. The invention also provides a sample processing apparatus adapted for use in the apparatus of the second aspect of the invention.
This invention is based on the development of a method of sample analysis and an apparatus for carrying out the method where samples are processed with one or more reagents and the products detected in a manner where the sample and reagents and products can be manipulated by movement along a conduit to various zones that can be heated in a controlled way and where the reaction products are detectable.
The method includes the steps of:
The method is useful for the analysis of a wide variety of sample types, especially where the sample must undergo one or more chemical transformations. The invention is most suited to the DNA analysis of samples, but is not limited to this use.
The sample may be introduced into the conduit by any suitable means. However, a preferred means is by using a free rolling ball held in a socket and rolling the ball across the surface from which the sample is to be obtained. A sampling device of this nature is the subject of the applicant's PCT patent application no. PCT/NZ04/0001 91.
The conduit has internal geometry that is preferably a single linear unbranched pathway and, although may be tapered, has no physical obstructions. However, the conduit may be non-linear or branched.
The sampling device may be constructed of any suitable material, but is preferably constructed from acrylic. The sampling device may alternatively be constructed from a hydrocarbon or perfluorocarbon plastic, a polymer resin, silicate (glass), silicon, doped silicon, or other semiconductor based materials, a metal, or a metal alloy. The ball is preferably constructed of the same material.
The conduit preferably has an internal surface that is coated, either prior to or during the course of sample processing, with an inert material such as a hydrocarbon, perfluorocarbon, or silicone wax, oil or grease.
The surface of the ball may be smooth, or may be textured or roughened, or coated with chemical modifiers, to minimise slippage of the ball on the surface when in use and/or to maximise or control sample adhesion.
Although a roller ball is the preferred means for obtaining a sample and delivering the sample to the conduit, other devices may be used, such as wheels, valves, vents, wicks, capillaries, syringes, pipettes, tweezers, probes, automated samplers, brushes, scalpels, needles, and fingers.
The sample may enter the conduit at atmospheric pressure, or under applied pressure, or by vacuum where the sample is sucked into the conduit.
The sample is preferably a laboratory prepared sample or biological material selected from the group including, but not limited to, blood, serum, saliva, urine, milk, and an extract obtained from bone, faeces, fat, flesh, hair, skin, plant material, microbes or microbial habitats. The sample may alternatively be a non-biological sample selected from the group including, but not limited to, water from waterways, industrial waste, and hazardous or non-hazardous chemicals including radioactive materials.
The one or more sample processing reagents may be any solid, fluid or gaseous reagent suitable for processing and/or analysis of the sample. In a preferred embodiment the one or more reagents are selected from the group, but not limited to hydrocarbon, perfluorocarbon or silicone greases waxes and oils; water based buffers and water solubilised organic and inorganic chemical reagents, trisma, magnesium chloride, potassium chloride, Bovine serum albumin (BSA), trehalose, ficoll, melizitose, sucrose, agarose; biochemical reagents required for enzymic DNA analysis including oligonucleotides, deoxynucleoside triphosphates and thermostable DNA polymerase.
Some or all of the one or more reagents may be hydrated or dehydrated by processes including, but not limited to, lyophilisation, evaporation, freeze drying or the like. For example, in the case of DNA analysis, the PCR reagents can be in the form of a lyophilised plug in the conduit of the sample device to maximize the stability of the reagents. During sample processing, the lyophilised plug is contacted with an aqueous solution causing the plug to rehydrate and be utilised in sample analysis.
In a preferred embodiment the conduit is open-ended allowing for pneumatic, hydrostatic, osmotic or electroosmotic control of samples, solutions of reagents and products, as well as for analytical interrogation.
The construction of the conduit may be by way of extrusion or pultrusion, such as in the formation of a tube, by machining (including drilling), by injection moulding, by injection compression moulding, by lithographic methods (including photon, electron or other particle based lithographies), by chemical etching, by contact or non-contact printing (including relief stamping), or by scratching, cutting or carving. As will be appreciated by those skilled in the art, manufacture of the apparatus of the invention is well suited to mass production, automation and miniaturisation.
The conduit passes through one or more temperature regulated zones and so facilitates temperature dependent processing of the sample and chemical reagents. The use of non-polar thermolabile chemical materials (such as waxes and greases) facilitates manipulation of the materials by changes in temperature to delay or initiate chemical activity within the lumen of the conduit. The appropriate arrangement of such materials enables long-term storage of prefabricated devices.
Surveillance and analysis of the conduit lumen can be performed by electronic (capacitive, inductive, resistive), optoelectronic (light scatter, refractive index, reflectance, absorbance, fluorescence, luminescence), ferro and para magnetic, resonance (nuclear magnetic, proton magnetic etc) methods. Such interrogation of the lumen can be performed transversely (through the lumen wall), axially (down the core of the lumen), or by a combination of both.
The processed sample can also be recovered from the lumen of the conduit for more detailed analysis. Such analysis could include chromatography, electrophoresis, spectroscopic techniques (mass, atomic, absorption etc), biological assay, macromolecular sequence analysis including nucleic acid and protein sequencing.
The invention has a number of important advantages. The fully self-contained chemistry requires no special technical knowledge by a user of the apparatus. No other materials, such as other reagents, are needed. The invention is well suited for use outside the laboratory environment. It is also easily adaptable to automation, including remote automation. The elimination of material transfer steps means that loss of sample is minimised.
The construction of the device is in two parts, the sampling device and the sample processing apparatus. This enables sophisticated processing and detection capability to reside in the sample processing apparatus while the sampling device, once used and contaminated with sample, can be disposable. The containment of sample processing reagents in the sampling device provides for an extended shelf life enabling ease of manufacture, distribution and storage. Further, the invention is amenable to miniaturization and mass production.
The invention also has the advantage of speed of sample processing. In the case of PCR analysis, the time taken from sampling to final result is typically less than 30 minutes
Other advantages will be apparent from reading this specification.
The invention will now be described by way of example with reference to
The cartridge device 1 comprises a sampling ball 2 housed within a socket 3. The ball 2 is free to rotate in any direction and has a surface that can range from substantially smooth to textured. The socket 3 is attached to or is integrally formed with the end of a shaft 4. The shaft 4 is tapered to facilitate easy entry to and exit from a receptacle 5 (as shown in
The shaft also serves as a handle. The shaft 4 may additionally be shaped or configured to suit the manner in which it is to be used, such as for holding by an operator during manual operation or for use by robotic manipulation.
The device 1 and the ball 2 may be constructed of any suitable material, typically acrylic or some other suitable plastics material.
The socket 3 comprises an opening 6 through which the ball is exposed and is accessible to a surface from which a sample may be obtained. The narrow gap between the ball 2 and the socket 3 represents the sample inlet 7. The diameter of the opening of the socket 3 is smaller than the diameter of the ball 2, thus preventing the ball 2 from falling out of the socket 3.
The inner wall 8 of the socket 3 opens behind the ball 2 to form a sample outlet 9, which is connected to the lumen 10 of the shaft 4. The lumen 10 extends the entire length of the shaft 4 and is a conduit through which the sample can be transported by applying reduced pressure (vacuum) or increased pressure to the lumen 10 at the distal end 11 of the shaft 4.
A series of plugs of paraffin 12 are located within the lumen 10. As will be appreciated by those skilled in the art, the plugs of paraffin 12 wet the inner surface of the lumen 10. The plugs of paraffin 12 form zones around an aliquot of aqueous material 13. It will be appreciated that paraffin can be used as a solid at room temperature, such as a wax or a grease, but as a fluid at elevated temperatures.
A lyophilised aliquot of reagents 14 is located within the lumen 10. For the purpose of PCR analysis, the lyophilised aliquot of reagents 14 contains a cocktail of biochemical reagents required including trisma, potassium chloride, magnesium chloride, Bovine serum albumin (BSA), trehalose, sucrose, melizitose, ficoll, oligonucleotides, deoxynucleoside triphosphates and thermostable DNA polymerase.
An aerosol filter 15 is also located within the lumen 10 at the distal end 11 of the shaft 4. The outer diameter of the aerosol filter 15 is such that it fits tightly within the lumen 10 and is held firmly. The aerosol filter 15 must be sufficiently porous to allow air to flow for the movement of samples and fluids within the lumen 10. The aerosol filter 15 is typically made from high molecular weight polypropylene.
Referring now to
The processing apparatus 16 also has three spatially separated heat zones 18, 19, and 20. The heat zones are in contact with the wall of the receptacle 5 and are able to transfer heat to the device 1 when inserted in the processing apparatus 16.
The heat zones 18, 19, and 20 may be constructed of any suitable material, but are preferably aluminium. The heat zones 18, 19, and 20 have embedded heating elements and temperature measuring elements to allow the setting, monitoring and control of the temperature of each of the heat zones 18, 19, and 20 independently from each other.
The heat zones 18, 19, and 20 are sufficiently spaced apart such that their temperature regulation is not coupled to each other. In other words, the temperature of one zone does not influence the temperature of another zone to any significant extent. The space may be any appropriate size, but is typically 3 mm.
For the processing of a sample containing DNA using PCR analysis, the heat zone 18 is set to 95° C. and is designated the denaturation zone. The heat zone 19 is set to 72° C. and is designated the extension zone. The heat zone 20 is set between the range 45-60° C. and is designated the annealing zone.
The heat zones 18, 19, and 20 will transfer heat to the receptacle 5 to create corresponding heat zones on the internal tapered wall 17 of the receptacle 5. Heat will then be transferred to the shaft 4 of the device 1 and to the lumen 10.
The heat zones 18, 19, and 20 are also fitted with a means to detect the presence of fluid plugs in the lumen 10. This may be by any suitable mechanism, but preferably by optoelectronic means.
The apparatus 16 also has a base unit 21, which houses a pneumatic pressure controller, a valve and a spectroscopic optoelectronic detector. The apparatus 16 also comprises, or is linked to, a microprocessor.
During operation the shaft 4 of the device 1 is held and the sampling ball 2 is rolled across the surface from which a sample containing the biological material of interest is to be analysed. Rotation of the ball 2 within the socket 3 causes sample to transfer from the ball 2 to the sample inlet 6. In addition, shear forces between the ball 2 and the socket 3 may help to facilitate disruption of cells in the sample.
The device 1 is then inserted into the receptacle 5 of the processing apparatus 16 as shown in
Firstly, the heat zones 18, 19, and 20 transfer heat to the lumen 10 of the shaft 4. The plugs of paraffin 12 then melt freeing the encapsulated aliquot of aqueous material 13 and enabling the lyophilised aliquot of reagents 14 to contact the freed aqueous material 13 and so rehydrate the lyophilised aliquot of reagents 14 to form a reagent mixture 22.
The steps 3 to 5 constitute a complete cycle of PCR and an effective doubling of the target DNA sequences. Any number of cycles may be used, but typically 35 cycles are used. Real time analysis is achieved during each cycle at the annealing stage.
For the purpose of PCR analysis, the lyophilised aliquot of reagents 14 contains trisma, potassium chloride, Bovine serum albumin (BSA), trehalose, oligonucleotides, deoxynucleoside triphosphates and thermostable DNA polymerase.
Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.
The method and apparatus of the invention are useful in a wide range of applications. The invention is particularly useful for the DNA testing of samples using PCR technology. However, the invention may be used for any sample processing where mixing with reagents is required before sample analysis. The invention has enhanced industrial application as it can be used out side the laboratory environment and does not need highly technical skilled operators.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NZ2004/000311 | 11/30/2004 | WO | 00 | 9/11/2007 |
