Technical field

The present invention relates to the preparation of samples for constituent analysis.

Background

Sample analysis has applications in many areas, for example the medical industry, diagnostics, and manufacturing. Samples are analysed to determine their constituent components. Samples requiring analysis are often fluids. One context is DNA analysis.

DNA analysis is particularly useful in medical diagnosis. For analysis of bacterial DNA, the DNA contained in bacterial cells and spores must be released through their rupture (lysis). It is known that DNA can be liberated from bacteria by mechanically vibrating a liquid sample containing the bacteria. However, this approach can take an undesirably long time.

Summary of invention

According to a first aspect of the present invention, there is provided a device for rupturing bacteria, the device comprising: a conductive body comprising a first face and a second face, wherein the first and second faces are separated by a gap; an input for introducing microwaves to the conductive body; and a channel for containing a sample; wherein: the conductive body is arranged to produce, upon application of microwaves at the input, parallel E-field lines extending in a first direction in a region of the gap; the channel runs in a second direction in the region; and the angle between the first and second directions is not more than 45 degrees. Thus, the invention provides a device for rupturing bacteria, in which efficient transfer of energy from the microwaves to a sample under analysis is not overly impeded by polarisation of the sample. This can reduce the time required to rupture bacteria within the sample.

In certain embodiments, the angle is not more than 10 degrees. This embodiment provides a greater efficiency of energy transfer to the sample, reducing the time required to rupture bacteria within the sample. In certain other embodiments, the angle is substantially zero. This orientation provides a particularly efficient transfer of energy to the sample, and minimises the time required to rupture bacteria within the sample.

There is also provided a method of rupturing bacteria using a device, the method comprising: applying microwaves to a conductive body via an input, the conductive body comprising a first face and a second face, wherein the first and second faces are separated by a gap and, due to the microwaves, parallel E-field lines extend in a first direction in a region of the gap; and inserting a sample into a channel, wherein the channel runs in a second direction in the region and, the angle between the first and second directions is not more than 45 degrees. Thus, the invention provides a method for rupturing bacteria, in which efficient transfer of energy from microwaves to a sample under analysis is not overly impeded by polarisation of the sample. This can reduce the time required to rupture bacteria within the sample.

Brief description of the drawings

For a better understanding of the present invention, and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the following drawings, in which:

Figure 1 is a cross- sectional but schematic view of a microwave device for sample preparation;

Figure 2 is a perspective but schematic view of the microwave device shown in Figure 1 ;

Figure 3 is a schematic view of an idealized, thin, cylindrical column of fluid positioned in, and at a first orientation to, a parallel electric field;

Figure 4 shows the same column as figure 3 but at a different orientation to the field;

Figure 5 shows the same column as figures 3 and 4 but at a different orientation to the field; Figure 6 shows a schematic representation of a microwave device for sample preparation wherein the sample channel does not pierce the conductive body; and

Figure 7 shows a schematic representation of a further microwave device for sample preparation.

Detailed description

Figures 1 and 2 show a microwave device 10 for sample preparation. The microwave device 10 includes a conductive body 11. The body 11 may be made from an electrically conductive material that may be magnetic or non-magnetic. The material may be metal, for example copper. The conductive body 11 is shaped into a split-ring shape.

The ring-shaped body 11 has a split in one side, which forms a gap 16. A first face 12 and a second face 13 of the conductive body 11 lie on either end of the ring and sit either side of the gap. The first and second faces 12 and 13 are flat and parallel. In other examples, the faces may be uneven, and may not be parallel. The conductive body 11 surrounds a hollow central cavity 19. The body 11 has, in the cross-section shown in Figure 2, a square outer edge. However, the body 11 could conceivably be made in any other regular shape (for example it could have a circular outer edge) or, indeed, the body 11 could have an irregular shape.

The device 10 has a port 14. Feed line 17 is conductively connected to the port 14 to apply microwaves to the conductive body 11. The microwaves are applied at a predetermined nominal frequency (though in practice the microwaves will have a certain bandwidth).

Upon the application of microwaves at the port 14, the conductive body 11 conducts an electrical current that oscillates when the frequency of the microwaves is at or around the natural resonant frequency of the split-ring body. The oscillating current in the body 11 creates an oscillating electric field E0 that extends between the faces 12 and 13 in much the same way that an alternating current source connected to a capacitor will establish an oscillating electric field between the plates of the capacitor. The electric field EO oscillates at the microwave resonant frequency. As shown, the port 14 is positioned on the body so as to couple energy from the microwaves from the feed line 17 into magnetic field of that is established in the cavity 19. The port 14 could be positioned differently on the body 11, however.

The electric field EO is shown by field lines 18, and the direction of the field is indicated by the direction of the arrow heads. The field lines are parallel across a region spanning the majority of the gap 16. The field lines run perpendicular to the first and second faces in that region. Although the electric field is parallel across the region, the field lines may bow out at the edges of the gap.

The device 10 has a tube 15 running through the conductive body 11. The tube 15 passes through the gap 16 and, as shown in Figures 1 and 2, through the first and second faces 12 and 13. The tube 15 defines a channel for containing a sample.

The tube 15 runs parallel to the direction of the field lines of the electric field EO. The tube 15 pierces the conductive body 11 such that the tube runs through the first face 12 and the second face 13. In Figure 1, the full length of the tube 15 in the gap 16 runs parallel to the field EO and perpendicular to the first and second faces 12 and 13. However, in other variants of the device 10, only part of the tube 15 may run parallel to the field E0 (for example, as shown in Figure 6).

When microwaves are applied to the conductive body 11 and an electric field is established in the gap 16, there is a transfer of energy from the electric field to the sample in the part of the tube 15 that lies in the gap 16. The amount of energy transferred to the sample depends on the strength of the electric field the sample is exposed to. The size and shape of the cavity 19 dictate the resonant frequency of the conductive body. When microwaves are applied at the resonant frequency of the conductive body, standing waves are formed in electric field in the gap 16, and energy transfer to the sample is enhanced. When the frequency of the microwaves is different to the resonant frequency of the split-ring body 11, the sample will experience much lower strength of electric field, and transfer of energy to the sample may occur but will be comparatively slight. There are two effects seen with energy transfer from a microwave electric field to a sample. The first effect is heating of the sample, which is caused by the presence of the electric field. Energy from the electric field transfers to the particles in the sample causing them to vibrate. Heating a sample containing DNA can denature DNA, which may not be desirable from the point of view of analysing DNA. Therefore, it is often desirable to avoid heating DNA. Limiting the amount of time the sample is exposed to the electric field can constrain heating of the sample.

The second type of energy transfer is known to chemists as the "microwave effect", and is the result of the sample being exposed to an electric field which oscillates at microwave frequency causing repetitive mechanical stresses on the particles of the sample. These mechanical stresses, when applied to bacteria, cause DNA to be liberated from the bacteria.

In order to liberate DNA from bacteria and yet not denature the liberated DNA through heating, the sample should be exposed to the microwave electric field in the gap 16 for a minimum amount of time. To transfer energy in a minimum amount of time the power transferred to the sample should be maximised.

The absorbed power P per unit volume (W/m ³ ) by a sample (assuming the sample is dielectric) is given by:

Ρ = σ£ ²

Where, σ is the effective conductivity (S/m) and E is the electric field strength within material.

Therefore the power absorbed by a sample is proportional to the square of the electric field experienced by the sample. To improve energy transfer to the sample, the strength of the electric field felt by the sample should be maximised. In the device 10 of Figures 1 and 2, the tube 15 filled with sample runs parallel to the electric field, which as will now be explained improves the efficiency of energy transfer from the electric field 18 to the sample. An effect called the depolarisation effect affects the strength of the electric field felt by the sample. The device 10 is designed to reduce the impact of the depolarisation effect.

The depolarisation effect is seen when a dielectric fluid interacts with an electric field. In device 10, a sample is placed in an electric field of strength E0. Dipolar particles within the sample align with the electric field in the gap 16 and polarize the sample. At any given moment, the alignment of the particles results in an excess of negative charges at the side of sample that is closest to the one of faces 12 and 13 that happens to be positively charged due to the oscillating current in the split-ring body 11 and an excess of positive charges form at the other one of faces 12 and 13 (which happens to be negatively charged at this time). In this sense, the part of the sample that is within the gap becomes polarised. These excess charges create a second electric field, which partially counteracts the external electric field E0 within the sample. The depolarisation reduces the strength of the overall E-field within the sample and therefore reduces the efficiency of energy transfer from the electric field to the sample. The depolarisation effect is particularly acute in aqueous samples owing to the large electric dipole moment of water, and bacterial samples to be analysed will normally be in water.

Figures 3 to 5 show the depolarisation effect on an idealized, thin, cylindrical liquid column subject to an applied parallel electric field. The applied electric field is the same in all three figures.

In Figure 3 the channel 20 is positioned perpendicular to the direction of the electric field E0. The sample is fluid and is in the channel 20, indicated by shading. Dipolar particles within the fluid align with the electric field. The aligned dipoles result in an excess of positive charges at a first side 35 of the column, and an excess of negative charges at a second, opposite side 36 of the column, shown in the figures as positive charges (+) or negative charges (-). The particles therefore polarize the fluid, and partially counteract the effect of the applied electric field. The two charge accumulations at the sides of the channel 35 and 36 create an internal electric field E _p inside the channel 20. The overall electric field experienced by the sample in the channel 20 is the sum of the external electric field EO and the internal field E _p . The fields EO and E _p work in opposite directions. It is desirable to maximise the electric field experienced by the sample in order to maximise the efficiency of energy transfer. To do this, the internal electric field E _p should be as small as possible.

The strength of an electric field at a distance r from a point charge of magnitude q is given by: ¾

Where k is a dimensionless constant. Therefore, to keep E _p small, the magnitude of each of the charge accumulations should be as small as possible, and the distance between the charges should be as large as possible. The magnitude of charge in the accumulations depends on the cross- sectional area of the sample which is perpendicular to the external electric field E0. The larger the cross- sectional area perpendicular to the field E0, the greater the magnitude of the charge that makes up the accumulations, and the stronger the electric field E _p is.

Therefore, decreasing the internal field E _p can be done by decreasing the cross- sectional area of the channel perpendicular to the electric field Eo and increasing the distance between the charge accumulations.

In Figure 3 the curved surface along the whole length 1 of the channel 20 (and therefore a corresponding surface of the sample) is perpendicular to the electric field E0. Further, the charge accumulations are separated by a relatively small distance (which is, at its greatest, the width w of the tube). A large cross-sectional area and small charge separation leads to a relatively large internal electric field E _p , which deleteriously counteracts the external electric field. Therefore the efficiency of energy transfer into the sample (e.g. for DNA liberation) in Figure 3 is relatively low. In Figure 4 the channel 20 is positioned at an acute angle a to the direction of the electric field. The angle of the channel relative to the electric field EO means that the cross-sectional area of the tube perpendicular to the electric field EO is smaller than that in Figure 3. There is also a larger distance between the opposing charge accumulations. Therefore the electric field E _p caused by the charge accumulations is smaller than that in Figure 3, so the sum of the external and internal electric field is larger (closer to the value of EO). The sample in Figure 4 experiences a greater electric field than the sample in Figure 3, and so energy is transferred to the sample more efficiently. In the case of rupturing bacteria, this results in a reduced time required to rupture the bacteria to liberate the DNA, and a reduced chance of heating the sample and denaturing the DNA.

Figure 5 shows the channel 20 aligned parallel to the electric field. In this arrangement, the charge accumulations arise at either end of the channel. Only a small cross-sectional area of the channel is perpendicular to the electric field.

Further, the distance between the charge accumulations is large. The charge accumulations here act, to an approximation, like point charges. The accumulated charges cause a very weak field in the direction opposing the electric field E0, and the electric field experienced by the sample in the tube is roughly the same as external electric field E0. The relatively high electric field experienced by the sample results in efficient energy transfer from the microwaves into sample.

Thus, the orientation of the channel in the device shown in Figures 1 and 2 effectively eliminates the impact of the depolarisation effect, such that energy transfer to a sample can be enhanced. However, improved energy transfer efficiency will be seen when the channel is positioned at any orientation other than perpendicular to the electric field. Therefore, variants of the device 10 of Figures 1 and 2 may have the channel positioned within 10° of the electric field lines, or within 20° of the electric field lines. The smaller the angle between the direction of the field lines and the direction of the channel, the higher the efficiency of energy transfer from the electric field to the sample. Figure 6 shows a further microwave device for sample preparation 10, similar to that shown in Figure 1 with like reference numerals indicating the same features.

Only part of the tube 15 in Figure 6 is parallel to the field lines of the electric field EO. The tube 15 does not pierce the conductive body 11, nor travel though the first face 12 and the second face 13. Instead the tube 15 travels into and out of the gap 16 from outside the conductive body 11, with a portion of the tube travelling parallel to the electric field EO. Other conceivable configurations of the tube 15, wherein a portion of the tube runs parallel to the direction of the electric field EO within the gap 16, could be used. The sample in the section of the tube 15 which runs parallel to the electric field EO experiences the strongest electric field as it is least affected by the depolarisation effect, and therefore sees more efficient energy transfer.

Another microwave device for sample preparation 40 is shown in Figure 7.

The device 40 comprises a conductive body 21. The conductive body 21 is spilt into two parts: a first part 22 and a second part 23. The two parts fit together to form a single hollow body defining a cavity 32.

The two parts 22 and 23 could fit together in a number of ways. The two parts 22 and 23 could clip together. Alternatively, the second part 23 may rest on top of the first part 22, or the two parts may be bolted together. Other conceivable methods of attaching the first part 22 to the second part 23 may be used.

When the first part 22 and the second part 23 are fitted together to form a single body, the parts are conductively connected. Therefore a current can flow between the first and second parts 22 and 23 without interruption.

The first part 22 of the body has a protrusion 25 which extends into the cavity 32. The protrusion forms a neck 24 in the cavity 32. In other words, the cavity 32 is a re-entrant cavity. The sides of the cavity facing each other across the neck 24 can be regarded as a first face 26 and second face 27 opposing each other across a gap. Of course, the protrusion 25 could equally well be provided on part 23 in order to define the neck 24. Also, it is of course possible to provide protrusions on both of parts 22 and 23, the protrusions extending towards each other to define the neck in the cavity 32.

In the device 40, the protrusion 25, the conductive body 21 and the cavity 32 are all cylindrical, and all share a common axis. However, the protrusion 24, the conductive body 21 and the cavity 32 may be other shapes, and may not share a common axis.

The device 40 has a port 29. A feed line 30 is attached to the port to apply microwaves to the conductive body 21. To ensure that there is adequate coupling between the feed line and the conductive body, the feed line extends a small way into the conductive body 21.

In Figure 7 the port 29 is positioned on the second part 23. Since the first and second parts 22 and 23 are joined together to form a single conductive body 21, they are conductively connected and the microwaves applied to the second part at the port 29 travel to the first part. Therefore the port can be positioned on either the first part 22 or the second part 23.

Upon application of microwaves to the conductive body 21, an alternating current, oscillating at the frequency of the applied microwaves, circulates within the body 21. Due to this alternating current, an electric field E0 is established between the faces 26 and 27 and across the neck 24. Over the majority of face 26 at the extremity of the protrusion 25, the electric field E0 extends perpendicularly between the faces 26 and 27. At the periphery of the protrusion 25 - its circumferential edge, recalling that it is cylindrical - the field lines of the electric field E0 will diverge somewhat. The microwaves can be applied at a resonant frequency of the body 21 at which a standing wave is established in the electric field E0 between the faces 26 and 27. When this condition is met, the transfer of the energy from the electric field E0 to the sample (e.g. to achieve lysis) is enhanced. At the same time, a resonant magnetic field is established in the cavity 32, and the port 29 is positioned to couple energy from the applied microwaves into this magnetic field.

The device 40 also includes a tube 31. The tube pierces the first part 22 and the second part 23 of the conductive body and traverses the neck 24. In a region of the neck 24 where the electric field lines are parallel, the tube 31 runs substantially parallel to the direction of the field lines. The tube 31 defines a channel in which a sample can flow. The tube 31 could be positioned differently in the conductive body 21 of Figure 7, such that it does not pierce the first and second faces 22 and 23 of the conductive body. In a similar arrangement to that shown in Figure 6, the tube 31 could have sections at different angles within the gap, such that only a portion of the channel is parallel to the electric field, but another portion is positioned at a different angle. In a further variant of the device of Figure 7, the tube 31 could be oriented so that it is not perpendicular to the faces 26 and 27. However, the further the tube 31 departs from the orientation where the tube is perpendicular to the faces 26 and 27, the more deleterious the polarisation effect will become. The conductive bodies illustrated in Figures 1, 2, 5 and 6 are straightforward and inexpensive to manufacture, requiring the shaping of a solid block of suitable material.

The microwave devices for sample preparation described above may be used to expose a large fluid sample to an electric field. In this case the fluid sample is moved through the tube, for example by pumping or through capillary action. As a volume of the sample moves through the tube, it is exposed to the electric field. At this time, energy is transferred to that volume of the sample. The volume is then moved further along the tube and out of the electric field. The sample is moved through the tube until all of the sample has been exposed to the electric field. The microwaves are pulsed constantly for the duration of moving fluid through the tube, which provides a pulsed electric field for the duration of the process. The microwave devices for sample preparation described above each have a single port for the application of microwaves to cause lysis. It is possible to add a second port to which a frequency sensitive detector can be coupled. That way, microwaves can be injected into one of the ports and extracted through the other port. Such an arrangement allows the resonant frequency of the device to be determined, e.g. by sweeping the frequency of the microwaves that are applied at one port and looking for a resonance in the microwaves that are extracted through the other port, e.g. by using a spectrum analyser as the frequency sensitive detector. The frequency of the microwaves that are applied to the device can then be tuned not to a nominal resonant frequency but to the actual, measured resonant frequency so as to better ensure an efficient transfer of energy to the sample that is to undergo lysis.

It must also be remembered that the drawings are schematic. For example, in practice, the internal and external diameters of tube 15 are typically 1 millimetre and 2 millimetres respectively and the perpendicular distance between faces 12 and 13 is typically 3-4 millimetres. Likewise, the internal and external diameters of tube 15 are typically 1 millimetre and 2 millimetres respectively and the perpendicular distance between faces 26 and 27 is also typically 4 millimetres. In the microwave devices for sample preparation described above, a sample is placed in, and orientated relative to, an electric field so as to decrease the impact of the depolarisation effect. Thus there are provided devices for sample preparation which achieve efficient energy transfer from an electric field. The devices provide a way of liberating DNA from bacteria in a relatively short amount of time without damaging the DNA through heating.

Those skilled in the art will appreciate that various amendments and alterations can be made to the embodiments described above without departing from the scope of the invention as defined in the claims appended hereto.