Patent Application
20130010567
The subject of the invention is an agitator of a liquid sample, which can be used to homogenise a solution by dispersing particles in it, heating the liquid, creating a nebulisation or imparting to the fluid energy sufficient to accomplish certain mechanical actions, such as the destruction (lysis) of such particles. Particles are taken to mean biological particles such as cells, bacteria, or again other particles, such as functionalised metal nanoshells.
A traditional method consists in subjecting the sample to ultrasound. This has a number of disadvantages, one of which is associated with the difficulty of transmitting sufficient energy to the sample. The sample is, indeed, enclosed in a tank, which may be a fine capillary, which is placed in the ultrasound tank filled with acoustic transmission liquid. Almost the entire energy in is lost due to the difficulty in establishing acoustic waves (pressure waves traversing the transmission liquid) with characteristics allowing satisfactory transmission of the energy to the liquid in the tank, notably limiting losses at the interfaces (the inner face and the outer face of the tank). Another disadvantage, which adds to the previous one, is that the energy which is nonetheless transmitted to the liquid in the tank does not necessarily produce the sought effect, i.e. heating, blending, dispersion, nebulisation, lysis or again rotation or centrifugation. Substantial energy must therefore be expended during a considerable period to accomplish satisfactory agitation. Blind application of mechanical vibrations, by positioning the tank on a vibrating membrane, for example, would have the same effects.
In addition, the current ultrasound blenders do not enable the agitation to be concentrated on a localised part of the sample. Focusing is taken to mean concentration of the vibrations stresses on a spatially restricted area.
Document FR-A-2 879 885 of the same inventor concerns a plate resonator which can be subjected to different modes of vibration by peripheral transducers, but which is envisaged above all as a microphone plate to transmit a vibration to the ambient air. The plate is made thinner in its centre, but is continuous, and it does not bear a load nor, above all, a tank. The bending modes which it is possible to impose on the plate are not themselves able to agitate an adjacent liquid sample.
The principal aim of the invention is to propose an agitator of liquid samples of satisfactory efficiency, i.e. which effectively transmits to the liquid in the tank a substantial proportion of the expended energy, and in such a way to enable genuine agitation of the sample, giving rise in it to appropriate movements or pressure variations.
In a general form the invention concerns an agitator of a liquid sample, including a tank containing the sample and a vibration generator, where the vibrations are communicated to the tank by an intermediate medium, characterised in that the intermediate medium is a solid ring resonator surrounding the tank and combined with the tank, and where the tank is attached to the centre of the ring and the vibration generator consists of at least one piezoelectric transducer installed in a peripheral region of the ring and constructed to transmit vibrations in the radial direction of the ring to the ring and to the tank.
Use is therefore made of vibrations in a solid communication medium to accomplish the agitation. The superiority of the invention lies in the fact that the vibrations are a mechanical displacement of large amplitude, which can exceed ten microns at 20 kHz, in a radial direction of the ring, i.e. parallel to its main plane. Such vibrations are particularly fit for producing the sought agitation. The transmission of such vibratory waves by the interfaces between the ring and the sample for agitation may be optimised to a high efficiency, such that the energy lost is relatively insubstantial. It is easy to optimise the installation to cause the ideal vibrations, notably by adjusting resonance frequencies over a preferential range between kHz and 70 kHz, and by exciting at these frequencies. Lastly, the use of a ring surrounding the tank concentrates or focuses the vibrations on the latter.
Main plane is taken to mean a plane perpendicular to the ring's axis of symmetry.
The tank will often be a tube with an axis which is roughly parallel with the ring's axis of symmetry perpendicular to the ring, and a part of which, for example the central part, will be surrounded by the ring. It will be seen that this arrangement enables a substantial agitation to be created, notably by a bending of the ring, leading to a bending of the tube, and notably of the part of the tube surrounded by the ring. This agitation can be extended throughout the content of the tube, although the vibratory waves arrive only in a small part of the tank and of the sample. These characteristics are obtained, for example, if the tube has opposite ends attached to a fixed frame belonging to the agitator.
Roughly parallel is taken to mean parallel to within ten degrees.
The resonator may be attached to the frame, notably, according to preferred possibilities, suspended from the latter by an elastic structure, or only suspended from the tube and independent of the frame.
A favourable installation consists in attaching the ends of the tube to the frame by supporting them on rubber discs (septums). It is possible to install interchangeable tubes in succession in the agitator and in the ring's central hole. Watertightness is maintained even if the ends of the tube are open. The frame may include holes revealing the rubber discs, which enables samples to be injected and then removed, using hollow needles which are sunk through the rubber discs. This arrangement allows facilitated integration in devices for treatment (preparation, analysis) of liquid samples, and notably in roaming devices, commonly designated by the term beacon.
It should be noted that the tube can either form a single piece with the ring, or be separate from it. It may be discarded after one use, which is convenient in the first case, or be washed before receiving a second sample, which may be preferred in the second case.
The agitation may be refined by creating a translation movement combined with a rotational movement of the fluid in the tube if the latter has an inner face with a helical twist, using a particular form of the vibrations.
Another improvement is accomplished if the ring tapers from the periphery to the tank, in order to concentrate the vibratory energy in a small volume of sample, and to impart to it the desired agitation firstly, before overall movements of the liquid diffuse it into the remainder of the tank.
According to another improvement the resonator includes, in addition to the ring, a central sleeve the axis of which is perpendicular to the ring in which the liquid tank is fitted. An axis perpendicular to the ring is taken to mean an axis which is roughly parallel with the ring's axis of symmetry. If the tank is tubular it can be made conical in shape in order to facilitate tightening. The sleeve lends itself to a satisfactory transmission of the vibrations to the content of the tank, and favours the application of the waves of vibration useful for agitation. In a preferred embodiment, it tapers from a place of connection to the ring until at least one end and, still more favourably, the sleeve is of generally equal thickness, either half the thickness of the ring where it is connected to the ring, or this thickness, depending on whether it transmits the vibrations to the tank by two free ends, or a single such end, and a zero thickness at the said end.
Another aspect of the invention is the choice of vibrations which are suited to agitation. It will be seen that there are several types of favourable vibration. Production of them by the ring does not pose any difficulties, due to the option of controlling them by a number of separate transducers, which are controlled separately. Be that as it may, the vibrations which it is principally envisaged to produce, and to apply to the sample, derive from a bending of the ring, i.e. the upper layers of the ring expand while the lower layers contract, and vice versa, leading to a substantial movement outside the plane of the ring at the place where the connection with the tank is made, transformed into a substantial compression movement, and therefore into a movement bending the wall of the tank at the end of the sleeve. These bending vibrations have the advantage that they have relatively low resonance frequencies, typically of the order of a few kHz to several tens of kHz, which are advantageous since they are accompanied by more ample movements. They can be obtained simply, by at least one transducer installed as a circle on the periphery of a face of the ring (upper face or lower face). Such bending vibrations can also be obtained by positioning such transducers facing one another on both faces, and controlled in opposing phases. They are transmitted efficiently to the sample and cause large-amplitude pressure waves with substantial mechanical effects.
The transducers can deliver different frequency vibrations. They can be positioned on both opposing faces of the ring to enable several simultaneous vibration controls. When the transducers follow the axial symmetry of the ring, for example fully covering the faces of the ring, the sleeve end vibrates, imparting a tank-tightening movement, which engenders a compression wave which increases as it moves inside the sample, reaching a maximum in the area of the central axis of this sample.
They can also be positioned in groups in respective sectors of arc on a peripheral region of the ring, delivering vibrations of the same frequency with phase offsets identical to angular offsets between the said circle sectors. This arrangement is used to create an overall rotating movement of the fluid due to a rotating tilting action of the sleeve which favours the blending of the fluid and, possibly, the diffusion of another effect of the vibrations throughout the entire sample.
One particularly appreciated embodiment includes firstly a complete circular transducer on one face of the ring to create an asymmetric vibration mode, in the form of a bending with axial symmetry, producing the compression waves in the sample, producing mechanical effects in it involving a first type of agitation; secondly, the group of piezoelectric transducers mentioned above on the opposite face of the ring to create a non-axisymmetric bending mode, and to impose a circular movement on the fluid involving a second type of agitation.
A magnet may be adjacent to the tube and to the ring in certain applications, for example to attach magnetic particles, for example metal nanoshells. The magnet then concentrates them in proximity to the ring, where they are subjected to the vibratory energy.
Several embodiments of the invention will now be described in detail in order to develop its various characteristic aspects, and the effects obtained. Reference will be made to the following appended figures, given purely as illustrations:
and
A first embodiment of the invention is represented in
The excitation of the transducers 15 and 16 deforms the resonator 11 and transmits vibrations to the tube 6 and to the sample 7. The mode of the vibrations is a bending of the ring 12 obtained by controlling the transducers 15 and 16 in antiphase, the transducer 15 expanding in the horizontal direction (radial of ring 12) when the opposite transducer 16 contracts. The shearing of the ring 12 due to the parallel and opposite movements of its lower and upper layers leads to bending movements in the centre, engendering a compression/expansion movement of the end of the sleeve 14, which is efficiently transmitted to the liquid sample. It has been observed that the excitation of the ring subject to bending gave relatively low resonance frequencies, which may be between several kilohertz and several tens of kilohertz, which have the advantage that they are accompanied by larger amplitudes and lesser damping: less electrical energy is therefore required to excite transducers 15 and 16. The application of vibrations in the radial direction (i.e. perpendicular to the resonator's axis of symmetry) perpendicular to the wall of the tube 6 using the sleeve 14 allows satisfactory transmission of the vibratory energy to the sample 7 through the interface constituted by the tube 6. An efficiency of 40% of energy transmitted (against 60% of energy reflected) may be attained when the tube is made from plastic, such as polypropylene. If there is no sleeve 14, the tube 6 would be subjected essentially to vertical vibrations, along its axis, which would produce only very little agitation of the sample 7, particularly since these vibrations would be almost entirely reflected at the interfaces of the tube. The energy transmission efficiency between transducers 15 and 16 and the ring 12 is also approximately 40%, if manufactured with care. The agitation produced by this energy arriving at the sample 7 is substantial since the tightening variations of the tube 6 by the sleeve modify its diameter and are transmitted to the sample 7 in the form of pressure waves creating the mechanical effects sought in the methods for destruction or fragmentation of particles, and possibly going so far as nebulisation or cavitation phenomena in the liquid, and involving substantial stirring of the sample 7, and therefore a blending of its parts. Moreover, the dissipation of the vibration energy in the area of the coupling of the sleeve 14 with the tube 6 can enable the sample 7 to be heated, or facilitate stirring due to the degassing in the liquid produced by the heating.
These effects are verified more specifically if the cavitation is obtained using quite ample movements of the tube 6. The thinning of the ring 12 towards its centre, making it more flexible in this location, increases the amplitude of the vibrations (by conservation of the quantity of movement) and concentrates the application of the energy whilst reducing the inherent frequencies.
The sleeve 14, which is required to apply a radial component element, also enables this energy to be distributed, over a sufficient volume of the sample 7 for its effect to be more emphatic, by avoiding too substantial a concentration. For example, it has been observed that by using a glass tube of outer diameter 2.5 mm, a coupling surface (surface of the sleeve in contact with the tube) of at least 3 mm2 allowed satisfactory agitation of a sample volume of several tens of mm3.
The resonator 11 may be made from aluminium or glass, notably thermally moulded or plastic (polypropylene, polycarbonate, etc.), the transducers 15 and 16 may be made from ceramics, and the tube 6 from metal, glass or plastic. The transducers 15 and 16 may be made from lead zirconate titanate or other piezoelectric materials such as barium titanate, or vinylidene polyfluoride (PVDF). Table I, in correlation with
The various settings are chosen so as to obtain relatively low resonance frequencies, of a few tens of kilohertz, in order to obtain more substantial movement amplitudes, typically between 1 and 20 microns of point-to-point amplitude in the area where the sleeve is coupled to the tube, and low damping. The audible frequencies will preferably be avoided, and this is the reason why resonance frequencies higher than 20 kHz will preferably be chosen. The resonance frequencies which will be sought are those of the ring 12, or of the unit constituted by the ring and the tube. It is possible in many cases to optimise the construction in order that one of these inherent frequencies is common to the ring 12 and to the tube 6. The two opposite transducers 15 and 16 enable the ring to be bent better using stress symmetry, but a single one might suffice.
They can be controlled at a single electrical frequency, or by a signal which is the superimposition (simultaneous application of several frequencies) or the juxtaposition (successive applications of signals of different frequencies during a predetermined time interval, for example of the order of one millisecond for each frequency component element), where these frequencies are normally chosen from among the resonance frequencies. They can be so chosen at the same frequencies, or on the contrary the excitation frequencies can be distributed between the transducers 15 and 16, since opposing excitations by the transducers 15 and 16 are not necessary. The ring 12 or the unit constituted by the ring 12 and the tube 6 each have several resonance frequencies, which correspond to the same number of separate inherent modes, but excitations at the first inherent modes (simple bending of the ring, with unvarying deflection from the periphery towards the centre, and bending of the tube 6 at two vibration nodes at the ends, and a single vibration antinode, at the connection to the sleeve 14, these modes being expressed as dotted lines in
Another embodiment will now be described by means of
Another embodiment is described in
Another difference from the previous embodiments is that the ring 212 is separate from the frame 201 and suspended only from the sleeve 214. This lack of a connection between the ring 212 and the frame 201 is beneficial because it prevents, even better than with the embodiment of
The sleeve preferably has sufficiently height for vibration nodes to appear. At design, the vibration profile of the sleeve will be observed, for example by laser vibrometry, and its height will be adapted such that both ends constitute vibration nodes. The adaptation of the height can also be accomplished by simulations.
A fourth embodiment is defined by means of
The first inherent mode of vibrations includes a single central antinode, and two nodes at the ends, as with the embodiments of
Compared to the first and second embodiments, the third and fourth embodiments enable a friction or a viscous damping between the sleeve and the tube to be prevented. This leads to more efficient transmission of the vibration energy to the sample results, together with less heating of the sample.
We now present a fifth embodiment of the invention, described by means of
The division of the transducers is necessary in this case due to the tightening screws 405 which must be avoided, but it must be stressed that this division gives a marked advantage: it enables vibration control to be applied with a phase offset, giving rise to a rotating movement of the sample 7.
Such a division of the resonator or at least of the transducers into sectors in order to apply phase-shifted excitations to obtain a circular movement of the tube is so favourable that it is envisaged to adopt it in other embodiments of the invention, notably those which have been previously described. It should be stipulated that, according to this embodiment, the resonator can remain monolithic and include multiple transducers on the same face, but it is preferable that the ring should also be divided into sectors, with the sectors of the ring preferably having the same angular openings as the transducers. Moreover, the tank tube is preferentially inserted by force in the sleeve in order that the vibrations traverse it, and do not cause it to rotate instead of the sample. Since the resonator is completely without the outline of the frame it is possible for the sectors to have 90° angular extensions when there are four such sectors, and for them to be separated by thin slits. A different number of sectors and phases can then be chosen, provided they number at least three to create the circular movement. The sectors 423 of the transducers 415 and 416 can be controlled either by the same piezoelectric polarisation and four electrical phases, as was suggested in connection with
Another aspect of the invention will now be described: the favourable dimensioning of the sleeve relative to the ring. The embodiment of
Similar dimensioning rules can be proposed for other embodiments. In that of
It is evident that other embodiments may easily be conceived from that which has been described, including by a substitution or by a combination of elements drawn from different embodiments.
The agitator may form a part of a more complete installation for treatment of samples of liquids, such biological liquids for example. The tank may be a tube commonly used in a biology laboratory, for example a traditional tube of Eppendorf or Falcon brand.
Throughout the description it has been shown how a device according to the invention enabled particles to be dispersed in a fluid. Of course, such a device also allows multiple liquids to be blended, for example immiscible or poorly miscible liquids.
Similarly, in the description, piezoelectric transducers have been described. In certain cases the invention can use other magnetostrictive transducers. Such transducers are electromechanical or magnetomechanical transducers.
However, piezoelectric transducers, and more specifically ferroelectric ceramics, are the preferred transducers.
We will now describe an example of the use of an agitator according to the invention for lysis of biological species such as spores, bacteria and viruses.
It is known that agitation of microballs in solution can enable lysis of such biological species present in the solution when the balls are agitated. Lysis is due to abrasive effect of the balls on the biological species. Microballs means balls with a diameter of less than 1 mm, typically a few ten to a few hundred μm.
The inventors attempted to use mobile agitators in the form of propellers, to cause sufficient abrasion of spores. They were confronted with a problem of reliability of such agitators. Furthermore, the presence of mobile parts immersed in the solution is a nuisance, for example they have to be cleaned between two uses. Furthermore such parts can wear mechanically.
By using an agitation device according to this invention, microballs can be entrained without having a mobile part immersed in the medium to be agitated. This also enables integration into a beacon.
One example device is given below: the actuator is composed of an aluminium resonator in the form of a large 50 mm diameter 2.65 mm thick ring 512 defining a 2.4 mm diameter opening at its centre
On each face of the ring (lower face and upper face), 4 identical lead zirconate and titanate type ceramic transducers 520 (made by Ferroperm—reference PT 26) arranged in four adjacent sectors of concentric rings. Each transducer has a large 25 mm radius, a small 10 mm radius and is 0.5 mm thick. Each transducer 520 on a particular face of a ring 512 has a phase shift of π/4relative to the adjacent sector. With this arrangement, each transducer on one face of the ring 512 is facing a transducer on the other face of the ring, two adjacent transducers being activated in phase opposition.
Each transducer 520 is excited by an AC signal with a peak to peak RMS voltage of 50V and RMS intensity 2.5 mA, and resonance frequency 20 kHz. The consumption of the device is equal to 500 mW, which corresponds to a power per unit volume of the liquid sample to be stirred (about 50 μl) equal to 10 W/cm3.
The liquid sample is dipped into a 34 mm high, 300 μm thick polypropylene tube 506 with a conical section, minimum outside diameter 2.4 mm, and maximum diameter 5 mm. 100 μm diameter zirconium dioxide (zirconia) balls are placed in the sample. Their mass is typically between 15 mg and 75 mg.
The polypropylene tube 506 is held in place by a coaxial aluminium sleeve 514. The sleeve 514 is fixed to the ring 512 and is coaxial with it. Its length is 3.8 mm. The inside surface of the sleeve 514 matches the outside surface of the tube 506. The tube 506 is held such that its bottom is 1.4 cm from the sleeve 514. Silicone grease may be placed on the outside surface of the tube at the contact with the sleeve, so as to facilitate transmission of the acoustic signal.
The liquid containing the spores may be water or a saline buffer. It may advantageously contain a lysis buffer which improves the efficiency of lysis, but this is not necessary.
Activation of transducers 520 generates a rotating bending wave, entraining microballs in the sample in a rotation movement. The microballs then cause abrasion of the spores making up the sample. The combined action of the rotating wave and rotation of microballs applies a rotation movement to the liquid sample, and a translation movement in tube 506. Consequently, most or even all of the sample is affected by abrasion by the microballs.
The spores containing the sample are Bacillus Subtilis and/or Bacillus Thurigensis type spores, and the concentration of the spores in the sample is 106 spores in 20 λl.
The agitation time is 2 minutes, which is sufficient to obtain a sample containing DNA of the lysed biological species, the lysis yield of the spores being nearly 100%. Such yields can be obtained with more complex commercially available devices with moving parts, and much more expensive. This device is also easily cleanable. The yield has been determined by quantification of purified DNA by a quantitative PCR type analysis.
This device may be integrated into a polycarbonate casing enabling electrical and mechanical confinement from the outside. The dimensions of this casing are included within a volume of 5 cm*5 cm*5 cm. The result is then a robust, nomad device that can be built into a beacon and consumes small quantities of energy.
Preferably, the device comprises a first needle 551 to convey the liquid sample, this first needle opening up into tube 506, for example at its top end. The device may also comprise a second needle 552, preferably opening up at the bottom end of the tube 506, and that will be used for sucking in the liquid sample contained in the tube. The first and second needles 551 and 552 may be made of metal, for example stainless steel, and may have a 450 μm outside diameter and a 350 μm inside diameter.
Preferably, the second needle 552 is centred in the tube 506. In other words, it is aligned with the longitudinal axis of the tube 506. Thus, the balls easily turn around the needle 552. The diameter of one end of the second needle 552 is less than the diameter of the balls so that the balls cannot be sucked in.
Thus, a device according to the invention can be used for lysis of biological species to be lysed. The device comprises:
Preferably, the phase shift between each transducer, considered to be in the same direction of rotation, has the same sign. When considering n transducers on a same face of the resonator, n≧2, the phase shift between two adjacent transducers may be 2π/N.
Preferably, the resonator comprises a projecting sleeve 514 coaxial with the centre of the ring 512, capable of holding the tube 506 containing the sample to be lysed in position.
Preferably, the device comprises a means 551 of conveying the liquid sample into the tube in the form of a first needle with an inside diameter between 10 μm and a few 100 μm. This first needle can open up in the upper part of the tube.
The device may also comprise a means 552 for sucking in the liquid sample contained in the tube, in the form of a second needle with an end diameter less than the diameter of the balls.
The lysis process using the device according to the invention then comprises the following steps:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 52000 | Mar 2010 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/054148 | 3/18/2011 | WO | 00 | 9/11/2012 |
