EP1075507A2

EP1075507A2 - Device and method for targeted exposure of a biological sample to sound waves

Info

Publication number: EP1075507A2
Application number: EP99927719A
Authority: EP
Inventors: Thomas Hahn; Bernhard Kleffner; Hans Ruf
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 1998-05-07
Filing date: 1999-04-23
Publication date: 2001-02-14
Also published as: US6699711B1; WO1999058637A3; DE19820466C2; DE19820466A1; WO1999058637A2

Abstract

Disclosed is a device for targeted exposure of a biological sample, preferably a biological cell material, to sound waves, comprising a sample container in which the biological sample is found in suspended form and an electroacoustic converter device generating the sound waves that is mounted outside the sample container so that the sound wave is coupled to the sample through the wall of the sample container. The invention is characterized in that the electroacoustic converter device generates sound waves at a frequency of at least 100 kHz, preferably 500 kHz to 5 MHz, and in that means are provided for focusing the sound waves which concentrate the sound waves in a sample volume inside the sample container of approximately 50 νl.

Description

Device and method for the targeted exposure of a biological sample to sound waves

Technical field

The invention relates to a device for the targeted application of a biological sample, preferably biological cell material or tissue samples, with sound waves with a sample vessel in which the biological sample is in suspended form, and with an electroacoustic transducer device which generates the sound waves and which is arranged outside the sample vessel is so that the sound wave is coupled into the sample through the sample vessel wall.

State of the art

A large number of applications of acoustic waves in the form of ultrasonic power sound for influencing material properties or a material structure are known. For example, ultrasound lithotriptors are used to smash kidney and gallstones using ultrasound. Ultrasound is also used for wound cleaning and phacoemulsification. In the non-medical field, ultrasound is used, for example, for cleaning, drilling, milling and welding.

The exposure of biological samples to ultrasound can produce certain biological effects, such as, for example, cell transport in cell reactors, which does not impair the vitality of the cells, or even destroy the cells, as is the case with the lithotriptors mentioned above. Due to the disintegrating effect on biological structures, ultrasound emulsifiers have gained considerable importance in sample digestion. Ultrasonic emulsifiers of this type, so-called sonotrodes, often have rapid transformers which are arranged on electroacoustic transducers, such as, for example, Langevinsche or compound transducers. The electroacoustic transducers often have a piezoceramic as the active material, and they vibrate with an amplitude of, for example, 0.5 μm. With the help of the transformers, the initially very small amplitude is multiplied to, for example, over 500 μm.

These ultrasonic emulsifiers have proven themselves in practice and are used frequently. The effect of the sound transmitted from the sonotrode to the biological sample depends on the sound pressure amplitude, which can vary greatly in space due to interference with sound waves reflected on the sample container. Since the temperature and the position of the sonotrode tip in the sample strongly influence interference phenomena and the sample usually heats up during treatment, it is difficult to precisely dose ultrasound for targeted excitation of biological effects.

Furthermore, immersing the sonotrode in the sample poses a risk of cross-contamination if the sonotrode is not cleaned with the necessary care when changing the sample.

The conventional design of the sound system does not prevent foam and aerosol formation, which reduce the reproducibility of the sound system.

Since the sonotrodes cannot be made arbitrarily small for electroacoustic reasons, there are limits to the treatment of miniaturized samples, as e.g. be used for genetic engineering analyzes during sample digestion.

DE 32 09 841 C2 shows an arrangement for embedding at least one tissue sample in paraffin. The device provides a container into which different coupling fluids can be introduced, in which a tissue sample to be treated is inserted. In order to accelerate the reactivity of the tissue sample with the respective liquids within the vessel, the device provides an ultrasound generator 61 on the bottom of the vessel, which couples ultrasound waves with a frequency of 35 kHz into the interior of the working vessel. With the selected frequencies, however, it is not possible to concentrate ultrasonic energy on the smallest sample volumes. The known device also provides an arrangement of the ultrasound-generating unit both outside and inside the sample vessel, which is why it can be assumed that the problem of cross-contamination does not exist in this case.

The desire for a concentrated entry of ultrasound waves in, for example, biological material is borne by the endeavor to bring about desired processes or reactions within the cell material. A typical example of such processes is cell disruption in order to examine the inside of the cells in further detection methods.

For cell disruption, use is made, as mentioned above, of, among other things, the use of ultrasound waves which, due to the biological cells present in a suspension, cause shear forces, as a result of which the cell walls are broken open. In order to improve the effect of cell disruption, ways have been labeled to increase the shear forces within the suspension. For example, macroscopic particles, preferably in the form of small glass beads, are additionally introduced into the suspension in order to increase the shear forces acting on the individual cells, which are responsible for cell breakdown.

In addition to the desire to increase the shear forces acting on the cells, there is also a need to miniaturize such devices, which appears to contradict the desire to increase shear forces. The invention has for its object to provide a device for subjecting a biological sample, preferably biological cell material, to sound waves, with which an exact intensity dosage of the sound waves is possible for targeted excitation of biological effects. Measures are also to be taken to increase the energy input based on the limited sample volume. In addition to the above requirements, it should in particular be possible to miniaturize the size of the device in order to be able to carry out a large number of sample examinations.

The object is achieved by a device having the feature of claim 1. Advantageous refinements are specified in the subclaims.

The device according to the invention for the targeted application of a biological sample, preferably biological cell material, with sound waves, with a sample vessel in which the biological sample is in suspended form, and with an electroacoustic transducer device which generates the sound waves and is arranged outside the sample vessel, so that the Sound wave coupling into the sample through the sample vessel wall is further developed such that the electroacoustic transducer device generates sound waves with a frequency of at least 100 kHz, preferably from 500 kHz to 5 MHz, and that means are provided for focusing the sound waves that the sound waves onto concentrate a sample volume within the sample vessel of approximately 50 μl.

In the ultrasonic device according to the invention, the electroacoustic transducer device is operatively connected to the sample vessel in such a way that the sound wave is coupled into the sample through the sample vessel wall and the electroacoustic transducer device is arranged outside the sample vessel. Since the sound is not radiated from the inside to the outside, as is the case with conventional ultrasonic emulsifiers in the sample, but is coupled in from the outside in, the sound intensity distribution can be restricted to the area of the sample, so that in conjunction with a higher center frequency, Loading range from more than 100 kHz, the influences can be controlled by interference of the sound waves reflected on the sample vessel wall. This allows an exact dosing of the sound intensities and precise excitation of certain biological effects in sample volumes down to 50 μl and below. Typical sample volumes in which biological effects are to be produced are approximately 100 μl.

In an advantageous embodiment, the sound is coupled in through the bottom of the sample vessel, so that the majority of the sound waves pass through the sample in a focused manner from below and are reflected at the upper boundary layer with reduced reflection on the wall of the sample vessel. This is an extremely effective arrangement for controlling disturbing interference phenomena.

In order to optimize the intensity coupling, a plane-parallel acoustic λ / 4-wave transformer is preferably provided in the sample vessel wall.

By inserting an acoustic lens into the sample vessel wall, the sound intensity distribution can be concentrated on a predetermined small volume area in the sample vessel. The acoustic lens is preferably designed as a spherical acoustic lens and can be inserted into the sample vessel base or forms it completely.

The sound wave is preferably coupled into the sample vessel by means of structure-borne noise, in that a soft polymer element or a fluid-containing coupling element is provided between the electroacoustic transducer device and the sample vessel and is in direct contact with both or a fluid coupling.

By the choice according to the invention of the ultrasound frequencies used of at least 100 kHz, preferably 500 kHz to 5 MHz, such high-frequency sound waves can be focused on the smallest areas, their spatial Sound intensity distribution can be influenced exactly. It is possible with such high-frequency sound waves to focus the area of the maximum sound pressure on the center of the sample volume, thereby avoiding spraying on the surface (aerosol formation) and thus cross-contamination between neighboring samples.

The sound coupling to the sample vessel wall by means of structure-borne noise is also essentially loss-free, so that even small transducer devices are sufficient to generate sound. Since they are arranged outside the sample vessel, they can also be of a very simple design in order to be able to provide the sound frequency required for the ultrasound treatment of biological samples. The ultrasound device according to the invention can therefore be miniaturized and can be used together with microtiter plate systems.

The sound wave coupling into the sample through the sample vessel wall is not subject to changing parameters, which makes it exactly reproducible. Precise reproducibility of the sound wave coupling is a prerequisite for precise dosing and automated sample processing, in which the sample vessel is automatically replaced or the biological sample in a sample vessel is automatically exchanged.

The invention is described below by way of example with reference to the drawings, without limitation of the general inventive concept. Show it:

1 shows an ultrasound device according to the invention in a perspective view together with a microtiter plate shown in the manner of an exploded view above the ultrasound device, FIG. 2 shows an electroacoustic transducer device which is used in the ultrasound device in FIG. 1, together with a section through a sample vessel of the microtiter plate from FIG. 1,

FIG. 3 shows an ultrasound device according to the invention with a piezoelectric composite, an electronic control device and a microtiter plate having several sample vessels,

4a and 4b show an SEM image of a piezoelectric composite and a schematic perspective illustration of the composite, and

Figure 5a to 5e, the manufacturing steps for producing a piezoelectric composite.

A first exemplary embodiment of a device for the targeted exposure of a biological sample, preferably biological cell material, to sound waves is shown in FIG. 1.

This device has a flat base plate 1, which is provided with a rail 2 on each of two opposite side edges. A crossmember 4 is mounted on the rails 2 and can be displaced in the longitudinal direction of the rail 2 (double arrow 5) and is driven by an electric motor (not shown) for displacement along the rail 2.

An electroacoustic transducer device 7 is displaceably arranged on the cross member 4 and can be moved back and forth along the longitudinal direction on the cross member 4 (double arrow 8) by a further electric motor (not shown).

On the side edges of the base plate 1 running parallel to the cross member 4, side walls 10 for receiving a microtiter plate 12 are provided. The micro Titer plate 12 is a plastic plate with several recesses arranged in a regular grid, each of which forms a sample vessel 13 for receiving chemical and biological and in particular microbiological samples. The microtiter plate 12 is placed on the device according to the invention, wherein it is carried by the side walls 10. The electroacoustic transducer device 7 can be moved in the plane of the base plate in the x and y directions, so that the electromagnetic transducer device 7 can be arranged under all sample vessels 13 of the microtiter plate 12. This electroacoustic transducer device 7 is shown in side view in FIG. 2 and has an approximately cylindrical body 14 made of a material that transmits sound well, such as a polymer, in which an electroacoustic transducer 15 is embedded. The electroacoustic transducer 15 is preferably made of a piezoelectric, magnetostrictive and / or electrostrictive material that can convert electromagnetic waves into sound waves.

The body 14 enclosing the electroacoustic transducer 15 is slidably attached to the cross member 4 by means of a holder 17.

It is also possible to fill the entire device with liquid and then to dispense with coupling cushions.

An acoustic lens 19 with a spherical surface 20 is arranged on the upward-facing end face of the body 14. With the aid of this acoustic lens 19, the sound waves emitted by the electroacoustic transducer 15 are bundled, as a result of which a sharply focused ultrasound beam is obtained.

A coupling element 22 for coupling the ultrasound beam into the sample vessel 13 is provided approximately in the center on the spherical surface 20 of the acoustic lens 19. The coupling element 22 consists, for example, of a soft, loss-free polymer or can consist of a thin-walled plastic vessel filled with a fluid suitable for sound transmission. One for the transfer of Fluid suitable for sound waves is, for example, water or preferably degassed water.

The crossmember 4 is preferably provided with a tilting device (not shown) with which the electroacoustic transducer 15 with the coupling element 22 can be raised and pressed against the sample vessel 13, the coupling element 22 being in direct contact with the vessel bottom 24.

The microtiter plates are preferably formed from polymer materials which transmit sound well, such as polystyrene, polypropylene or polyethylene, the contact areas of the vessels 13 with the electroacoustic transducer device 7 - in the illustrated embodiment these being the vessel bottoms 24 - preferably being membrane-like, or in particular plane-parallel acoustic λ / 4 - Form shaft transformers. However, it is also possible within the scope of the invention to design the vessel bottom 24 of the sample vessel 13 as an acoustic lens with a spherical surface instead of the acoustic lens 19 arranged in the electroacoustic transducer device 7.

It is essential for the invention that the sound wave coupling into the sample contained in the sample vessel 13 takes place through the sample vessel wall, so that the sound is coupled in by means of structure-borne noise in exactly defined conditions. This coupling by means of structure-borne noise has low losses, so that the sound intensity required for the treatment of the sample materials can be provided even with small electroacoustic transducers. By focusing by means of the acoustic lens 19, the sound waves are concentrated on the sample material, so that the sound wave energy is used very effectively. In addition, the electroacoustic transducer device 7 according to the invention does not come into direct contact with the sample material, so that it can be moved quickly between individual sample vessels 13 of a microtiter plate 12 without the risk of contamination. Because of the very effective coupling, the intensities generated by the converter 15 can be relatively small, the electro- acoustic transducer 15 can be miniaturized. Since the transducer does not come into contact with the sample material during coupling and can therefore be moved quickly between individual sample vessels 13 without an additional cleaning process, the ultrasound device according to the invention is ideally suited for automated ultrasound treatment of a large number of samples.

High-frequency ultrasound with a frequency of at least more than 100 kHz and in particular 500 kHz to 5 MHz is used as the ultrasound, since high-frequency sound can be focused on the smallest volumes, so that even sample volumes of approximately 50 μl can be sonicated separately. The maximum sound pressure can be focused in the middle of the sample volume, as a result of which spraying on the surface (aerosol formation) and thus cross-contamination between sample materials of adjacent sample vessels 13 are avoided.

A further exemplary embodiment according to the invention of an ultrasound device for the targeted exposure of a biological sample to sound waves is shown schematically in FIGS. 3 to 5e. As an electroacoustic transducer device 7, this ultrasound device has a so-called piezoelectric 1-3 composite. In such a piezoelectric 1-3 composite, a multiplicity of individual sound transducers 30, which are each formed from a piezoelectric rod 35, are arranged in a regular, for example, square grid, their longitudinal extension being oriented perpendicular to the grid plane. These individual sound transducers 30 are embedded in a polymer matrix 31 for mutual isolation and for transmission of the sound generated (FIGS. 4a, 4b). Composites of this type are produced by the dice and fill process, with plane-parallel disks 33 being first cut out from a cuboid block of piezoelectric material on a base area 34 (FIG. 5b), and disks 33 in the next working step in the transverse direction to the first cutting direction can be cut into rods 35 standing on the base 34. These rods 35 are cast together with the base 34 into the polymer matrix 31 (FIG. 5d). Finally, the base 34 is worked off, for example by grinding. processes so that only the rods 35 remain as individual sound transducers 30 in the polymer matrix 31 and are connected to corresponding contact elements for the application of an electrical frequency signal. It is also possible to structure this electrode in the form of individual elements (ultrasound array).

An electrical control device for controlling the individual sound transducers 30 has a frequency generator 40, a phase shifter device 41 and a phase control device 42. The frequency generator 40 generates an electrical frequency signal with a frequency of at least 100 kHz and in particular 500 kHz to 5 MHz. This frequency signal is fed into the phase shifter device 41, which has a multiplicity of individual phase shifters, with which the phase of the incoming frequency signal can be changed, the phase shifter device 41 having a separate output for each individual sound transducer 30, so that one for each individual sound transducer 30 the other individual sound transducers 30 independent phase signal can be output. The individual phase shifters are controlled by a phase control device 42.

The predetermined phase differences of the individual sound transducers 30 result in controllable constructive and destructive interferences of the sound waves generated, as a result of which exact focusing is achieved without additional mechanical focusing means and a very precise three-dimensional intensity distribution is obtained. The focus can also be changed as quickly as desired. A thin, disk-shaped coupling element 22 made of a soft, loss-free polymer or a plastic body filled with a fluid is preferably arranged on the electroacoustic transducer device 7. If the individual sound transducers 30 embedded in a polymer matrix 31 are used, the polymer matrix 31 itself can be used as a coupling element, that is to say that the individual sample vessels 13 are placed directly on the electroacoustic transducer device 7 designed as a composite. A fluid as a coupling layer is also possible. The electronic control of the individual sound transducers 30 arranged in a two-dimensional array permits both sequential and parallel sonication of the individual sample vessels 13. This array arrangement of the individual sound transducers 30 also enables a further miniaturization of the ultrasound device with ideal, arbitrarily adjustable and rapidly changing intensity distributions , so that even complicated methods for applying samples to ultrasonic waves can be carried out simply, quickly and, above all, automatically.

The piezoelectric composites are particularly advantageous for the device according to the invention, since they allow effective, broadband sound generation and coupling, simple further processing and extensive adjustment of the electroacoustic properties. However, such array arrangements can also be produced in a conventional manner from piezoceramic elements or lead zirconate titanate elements, the electronics for controlling them being essentially the same.

A major advantage of the array arrangement is that a large number of samples, which are located in the sample vessels of a microtiter plate or another cartridge system, can be used with ultrasound sequentially or in parallel or in any other order without a relative movement between the sample vessels and the electroacoustic transducer device can be sonicated. Since no relative movement is necessary, the sample vessels do not have to be moved when treating multiple samples, which avoids cross-contamination, and it is not necessary to mechanically couple the electroacoustic transducer device to a sample vessel each time anew, which must be done very carefully to ensure that the to obtain the desired, reproducible coupling states.

In summary, it can be stated that the invention allows an exact, reproducible coupling of the sound waves into sample vessels, whereby the in the field intensities generated in the sample vessels can be precisely adjusted and metered. This allows specific chemical, biological or microbiological effects in the samples to be specifically stimulated. In addition, interference effects can be reduced or completely avoided by focusing on the smallest sample volumes.

REFERENCE SIGN LIST

baseplate

rail

traverse

Double arrow electroacoustic transducer device

Double arrow

Side wall

Microtiter plate

Sample vessel

Body of electro-acoustic transducer

Bracket acoustic lens spherical surface

Coupling element

Vessel bottom

Single sound transducer

Polymer matrix

Slices

Floor space

rod

Frequency generator

Phase shifter device

Phase control device

Claims

P ate nta nsp rü ch e

1. Device for the targeted application of a biological sample, preferably biological cell material, with sound waves, with a sample vessel (13) in which the biological sample is in suspended form, and with an electroacoustic transducer device (7) that generates the sound waves, which is outside the sample vessel (13) is arranged so that the sound wave is coupled into the sample through the sample vessel wall, characterized in that the electroacoustic transducer device (7) generates sound waves with a frequency of at least 100 kHz, preferably from 500 kHz to 5 MHz, and that means are provided for focusing the sound waves, which concentrate the sound waves on a sample volume within the sample vessel of approximately 50 μl.

2. Device according to claim 1, characterized in that the electroacoustic transducer device (7) is provided below the sample vessel bottom (24) and the sound wave coupling takes place through the sample vessel bottom (24).

3. Apparatus according to claim 2, characterized in that at least the sample vessel bottom (24) consists of acoustically suitable materials such as polystyrene, polypropylene or polyethylene.

4. Apparatus according to claim 2 or 3, characterized in that at least the sample vessel bottom (24) is membrane-like.

5. Device according to one of claims 1 to 4, characterized in that a plane-parallel aku- Stical λ / 4-wave transformer is provided in the sample vessel wall.

6. Device according to one of claims 1 to 5, characterized in that the means for focusing the sound waves is a spherical acoustic lens which is inserted into the sample vessel wall.

7. Device according to one of claims 1 to 6, characterized in that a soft polymer element can be attached to the electroacoustic transducer device (7) for coupling sound waves to the sample vessel (13).

8. Device according to one of claims 1 to 6, characterized in that for coupling sound waves to the sample vessel (13) a fluid-containing coupling element (22) on the electroacoustic transducer device (7) can be used.

9. The device according to claim 8, characterized in that the fluid of the coupling element (22) is degassed water or another liquid which transmits sound.

10. Device according to one of claims 1 to 9, characterized in that the electroacoustic transducer device (7) has an electroacoustic transducer (15; 30) formed from a piezoelectric, magnetostrictive and / or electrostrictive material.

11. The device according to claim 10, characterized in that the means for focusing are designed such that the sample volume into which the sound waves can be focused, within the sample vessel (13) is adjustable at a distance from an average level.

12. Device according to one of claims 10 to 11, characterized in that the electroacoustic transducer device (7) has a plurality of electroacoustic transducer elements (30), a control device being provided as a means for focusing, which is designed such that the transducer elements (30) are controlled with predetermined phase differences, so that there is a predetermined focus due to constructive and destructive interference of the sound waves generated.

13. The apparatus according to claim 12, characterized in that the transducer elements (30) are arranged regularly distributed in a 2-dimensional arrangement.

14. Device according to one of claims 1 to 13, characterized in that the transducer device (7) is formed from a Piezokompo- site (30, 31) which as a learning elements (30) several in a polymer matrix (31) embedded piezo elements ( 30).

15. The apparatus according to claim 14, characterized in that the piezo elements (30) are rod-shaped and are arranged in a regular grid in the matrix (31).

16. The apparatus according to claim 14 or 15, characterized in that the piezo elements (30) are arranged in a matrix, the rod-shaped piezo elements (30) being aligned with their longitudinal axes perpendicular to the lattice plane.

17. Device according to one of claims 1 to 16, characterized in that the electroacoustic transducer device (7) for the automatic treatment of several samples is arranged on an XY slide which can be moved in one plane.

18. Device according to one of claims 1 to 17, characterized in that the sample vessel (13) is part of a microtiter plate with a plurality of sample vessels (13).

19. Device according to one of claims 1 to 18, characterized in that the sound coupling from the transducer is carried out by a fluid in the sample vessel wall.

20. A method for the treatment of biological samples by means of sound waves with a device according to one of claims 1 to 19, characterized in that several samples are automatically sonicated.

21. The method according to claim 20, characterized in that a device according to claims 1 to 12 is coupled with its electroacoustic transducer device to a sample vessel arrangement with a plurality of sample vessels, and the samples located in the sample vessels without a relative movement between the sample vessel arrangement and the transducer device in series or be sonicated in parallel.