GB2403729A - Sonicator device and method - Google Patents

Abstract

A sonicator device, especially a micro-sonicator (2) comprises a lysis chamber (4) covered by a membrane (8). An ultrasonic driving means such a piezoelectric transducer (14) is provided on the membrane (8) to vibrate it at a driving frequency. The device has a monitoring means (40) for monitoring the performance of the sonicator device. A control unit (42), responsive to the output from the monitoring means (40), determines the resonant frequency for the device and controls a drive unit (44) to drive the sonicator device at the determined resonant frequency. The control means (42) is adapted to monitor any changes in the resonant frequency of the device due to different characteristics in the chamber (4) and adjust the driving frequency accordingly and therefore effectively comprises a feedback control for giving real-time control of the optimal driving frequency.

Description

Sonicator Device and Method This invention relates to sonication devices,

especially micro-sonicators, for cell Iysis or disruption of biological material, e.g. bacterial spores, more particularly to a micro sonicator device having an ultrasonic driving unit operating at the resonant frequency of the sonicator chamber and its contents and more particularly to a micro-sonicator which is part of an in-line sampling apparatus.

The first stage of a biodetection system often involves pretreating a sample so as to improve its detectability. For example, Polymerase Chain Reaction (PCR) enables replication of DNA contained within a sample. Thus for PCR the DNA must first be extracted from the material of interest. The first stage of this involves breaking open the cells or the disruption of the spore structure in such a way as to free the DNA from within.

This disruption process is termed Iysis specifically for cells but within this document will also refer to the process by which spores are disrupted.

It can often be advantageous to modify the surface characteristics of samples such as bacterial spores. For instance immunoassays involve the binding of an analyte to a specific antibody contained on the surface of a sensor. Detection sensitivity can be improved by modification of the surface of the species to be detected so as to improve subsequent binding to the antibodies on the biosensor. For the purposes of this specification modification of the surface of spores for the purpose of pretreating to aid subsequent detection shall be deemed to fall within the term Iysis.

Cell Iysis can be achieved in many ways. The methods may be chemical or physical.

Chemical methods, such as addition of detergent to weaken the cell wall, suffer from the disadvantage that once Iysis is achieved it is often necessary to clean the Iysed cells of the Iysis solution. This adds additional steps and, in a flow through fluidic system complicates the system required.

Macro scale physical methods are also known. Sonication involves the introduction of a vibrating probe into the suspension of cells. This method has several drawbacks however - the method is difficult to control and cells Iysed early in the process tend to become further degraded later in the process. Further the method tends to introduce local heating of the sample which can degrade the material of interest. Also sonication on such a macro scale generally requires relatively large samples and consumes considerable power and is not suitable for flow through systems. Further for systems that are to be used for in situ monitoring in the field macro scale sonication is not ideal.

US Patent 6,100,084 describes a micro-sonicator device. A small Iysis chamber is formed using micro-machining techniques and provided with an ultrasonic transmission medium. A membrane closing the chamber has a piezoelectric layer disposed thereon.

The piezoelectric material is driven by an AC voltage source which causes the membrane to vibrate at the frequency of the driving source thus exciting the ultrasonic transmission medium and causing Iysis of a cell or spore disposed therein. The device described in US6,100,084 is not a flow through system however. Flow through systems are advantageous for performing various different assays in a simple and speedy fashion.

US Patent 6,440,725 describes a microfluidic flow through system for processing fluid samples. The apparatus described can include a flow through Iysis chamber having an ultrasonic horn coupled to the chamber for transferring ultrasonic energy to the fluid sample in the Iysing region at the resonant frequency of the cells. It is noted however that the conditions in a flow through chamber can vary however and Iysis may not always be as efficient as possible.

It is therefore an object of the present invention to provide an improved sonication device for cell or spore Iysis.

Thus according to the present invention there is provided a sonicator device comprising a chamber formed, at least in part, by a flexible membrane and having an ultrasonic driving means mounted on said membrane characterized in further comprising a monitoring means, for monitoring the performance of the sonicator device and a control unit, responsive to said monitoring means, to allow, in use, the ultrasonic driving means to operate at the resonant frequency of the device.

The present invention has a control means, essentially a feedback control methodology, to ensure that in use the device can be operated at the resonant frequency of the sonicator device. Operating at the resonant frequency of the device ensures that energy is coupled into the device most efficiently and the more efficiently energy is coupled into the device the greater the efficiency of Iysis. The resonant frequency of the device will depend however upon the nature of the sample within the chamber. Further the

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resonant frequency of the device can change during operation. The introduction of bubbles into the chamber, which can occur during filling, will alter the resonant frequency which can have severe effects on the operation of Iysis. The present invention therefore uses a monitoring means to measure the resonant frequency of the device and a control means therefore ensures, when Iysis is required, that resonance is maintained by altering the driving frequency of the driving means accordingly. Thus real-time efficiency control is achieved.

Preferably the chamber is provided with at least one inlet channel and/or at least one outlet channel. By providing inlet and/or outlet channels, preferably both, the chamber can be formed as part of a micro-fluidic system. This allows for preparation of the sample prior to Iysis, or post Iysis treatment. Certain reagents could be added to the chamber before, during or after Iysis or the sample could be taken from the Iysis chamber for post processing or for various assays to be performed. The output channel may be conveniently arranged to be vertical so that any bubbles present in the chamber float out of the chamber. As will be described later the presence of bubbles can be a major factor in changing the resonant frequency of the chamber. Indeed a sonication chamber with a vertically arranged channel could possibly be operated at a fixed resonance, i.e. without the feedback control.

Conveniently the sonicator chamber is formed in part from a semiconductor material.

Semiconductor materials can be processed using various micro-machining techniques to form desired structures which can be very small and intricate. Conveniently the sonicator chamber could be formed as a cavity in the semiconductor material by any usual technique such as etching. Silicon is a suitable material as it is highly machinable using MEMS fabrication techniques although any rigid material, e.g. metal or glass would suffice. Silicon is also useful when the device is part of an integrated system. The other components of the system could also be made out of silicon and the entire system could be fabricated at the same time using well known processing techniques. In an integrated system there may well be other advantages in using silicon. For example there is a well defined chemistry for trapping DNA on the surface of oxidised silicon (silicon dioxide).

Silicon, and other semiconductors can also have a high thermal conductivity. Therefore the chamber can be coupled to a cold source to remove any heat generated during operation. Unwanted heat can cause unwanted degradation of the Iysis products. In any case use at the resonant frequency efficiently couples ultrasound energy into the device so unwanted heating effects may be minimised.

Where the chamber has at least one input and/or at least one output channel these could also be formed in the semiconductor material. Indeed semiconductor processing can be used to form a micro-fluidic system with various components to produce very small and efficient sampling devices so called 'labs-on-a-chip'.

The membrane is conveniently formed from glass. Glass is a cheap and easy material to use and has both stiffness and flexibility. Indeed use of both a glass membrane and a semiconductor, especially silicon, chamber results in a sonicator that has a high quality, or Q. factor. A high Q factor means that the device will exhibit a strongly resonant behaviour. At the resonant frequency energy will be very efficiently coupled into the system. However away from the resonant frequency the coupling will be increasingly less efficient. The present invention can operate with devices with a high quality, or Q. factor to achieve efficient coupling at the resonant frequency and, by monitoring the resonant frequency, prevent inefficient operation when conditions in the chamber change the resonant frequency thereof. A sonicator device with a relatively high Q factor is therefore preferred. The skilled person will understand that the Q-factor for the system as a whole could be considered as the resonant frequency divided by the FWHM (Full Width at Half Maximum - the difference between the frequencies on either side of the resonant frequency where the output is 50% of that at the resonant frequency). The measured output may be the impedance or the acoustic output over the frequency range.

For the purposes of this specification a high Q factor is taken to mean a high Q factor when filled with air and a high Q factor is taken as being greater than 15, greater than 20, greater than 25 or greater than 30.

The ultrasonic driving means may be anything that can supply ultrasonic energy to the chamber via the membrane, for instance an ultrasonic horn. Conveniently though the ultrasonic driving means comprises a layer of piezoelectric material disposed on the membrane. Supply of an AC voltage to the piezoelectric material causes it to vibrate thereby causing vibration of the membrane. Use of piezoelectric material offers a simple driving means that can be easily bonded to the membrane and sized accordingly.

Piezoelectric material could be deposited onto the membrane and formed into transducers or transducers could be bonded to the membrane. PZT (lead zirconium titanate) is a suitable piezoelectric material.

The monitoring means may comprise an acoustic sensor, such as a microphone located adjacent the chamber. As will be understood at the resonant frequency more energy is being coupled to the chamber so the acoustic output thereof will be greater. Thus by measuring the acoustic output across a range of driving frequencies the resonant frequency can be detected as would be understood by one skilled in the art.

The resonant frequency can also be determined by looking at the electrical impedance of the sonicator. The impedance will be lowest at the resonant frequency and hence the monitoring means may comprise a means responsive to the impedance of the device.

One convenient way of determining the impedance is to measure the current that the sonicator draws which will be greatest at the resonant frequency. Therefore measurement of the current through a resistor electrically connected to the sonicator can be used to give an indication of the resonant frequency. Determining the impedance of the device or simply measuring the current through a resistor gives a very simple method of determining the resonant frequency without requiring additional sensors and therefore is particularly advantageous. Obviously the current through the resistor is related to the voltage across it so measurement of the voltage also gives a convenient way of determining the current through it and hence the impedance.

Other suitable monitoring means will be apparent to the skilled person. For instance the monitoring means could measure the vibration of the membrane, say through use of a laser vibrometer or strain sensor, to determine the frequency of greatest amplitude vibration.

Alternatively, given that Iysis occurs to the greatest extent at the resonant frequency a direct in-situ measurement of Iysis could be used. For instance chemical reagents that react with the products of Iysis to produce light could be added to the chamber. The monitoring means could then comprise a photodetector to monitor the light output from the chamber.

Alternatively sonoluminescence could be used as an indicator. Sonoluminescence, as will be understood by one skilled in the art, is a phenomenon whereby sonic stimulation of a liquid can give rise to luminescence. The intensity of luminescence could then be used as a measure of efficiency of energy transfer to the chamber and hence resonant frequency. Again then the monitoring means may comprise a photodetector to monitor the light output from the chamber.

The control means could conveniently comprise a processor. As used herein the term processor could comprise a suitably programmed computer, a microprocessor, an FPGA array or any dedicated circuitry. Use of a programmable processor is preferred as it allows various control regimes which may be optimised for different situations. The control means controls the driving frequency of the ultrasonic driving means and is responsive to the monitoring means to ensure that the resonant frequency is maintained.

For instance the control means may be adapted to periodically scan the ultrasonic driving means across a frequency range and determine the resonant frequency from the output of the monitoring means. By operating the driving means at different frequencies within a range, which could be a relatively broad range in which the resonant frequency is expected to lie or a narrow range based on a previous measurement, the monitoring means can measure the performance, e.g. acoustic output, across this range of frequencies. Based on the output from the monitoring means the resonant frequency can be determined, e.g. the frequency giving rise to the greatest acoustic output, and that frequency maintained until the next scan.

In an alternative method of operation which could be complementary the resonant frequency could be determined and the value of the output of the monitoring means noted. That frequency of operation could then be maintained unless the output from the monitoring means changes by a certain amount. The skilled person would be aware of a large number of possible scanning routines or algorithms that could be applied to ensure that the resonant frequency is maintained.

It should be noted however that whilst resonance is preferred for Iysis there may be reasons why one would wish to operate the device off resonance. For instance it may be desired to mix the contents of the chamber such as various reagents. Alternatively certain reactions may be stimulated by supplying ultrasonic energy to the chamber but not such that additional Iysis is achieved. Therefore the control means may be adapted to track the resonant frequency and operate at resonance when Iysis is intended but operate a different frequencies for different purposes.

The device may also be provided with more than one ultrasonic transducer. Certain sonochemistry reactions are optimised when performed in a chamber excited by two transducers operating at different frequencies. The control means may control both devices according to the intended purpose.

The sonicator is preferably a microsonicator, i.e. one having a chamber with a volume of less than 10 pl or less than 5,ul or less than 1 Saul.

In a second aspect of the invention there is provided a method of sonicating a sample comprising the steps of; i) introducing a sample to a chamber comprising, at least in part a flexible membrane connected to an ultrasonic driving means, ii) determining the resonant frequency of the chamber, and iii) driving the ultrasonic driving means at the determined resonant frequency. The term sonicating shall be taken to mean, for the purposes of this specification, treating a sample with ultrasonic energy, e.g. for cell or spore Iysis.

The method according to the second aspect of the invention therefore provides an efficient way of achieving cell or spore Iysis by ensuring that the driving means is operated at the resonant frequency of the device. All the advantages and embodiments of the apparatus described above with respect to the first aspect of the invention are applicable to the method of the second aspect of the invention.

In particular the step of determining the resonant frequency of the chamber may comprise the steps of driving the ultrasonic driving means across a range of frequencies and monitoring a characteristic of the chamber which has a turning point at the resonant frequency. In other words a characteristic is monitored which has a measurable value which is a maximum or a minimum at the resonant frequency.

The characteristic measured may be acoustic output from the chamber in which case the step of monitoring the acoustic output comprises providing an acoustic sensor adjacent the chamber. Alternatively the characteristic measured may be related to the impedance of the ultrasonic driving means, such as the current through a resistor connected to the ultrasonic driving means.

According to a third aspect of the invention there is provided a control apparatus for a sonicator device comprising a monitoring means for monitoring the performance of the sonicator device and a control unit, responsive to said monitoring means, for ensuring that, in use, the sonicator device operates at its resonant frequency. A control apparatus according to the present invention may be applied to a sonicator to provide efficient operation thereof.

The sonicator device as described above may be especially usefully employed in sonochemistry, a branch of chemistry in which reactions are stimulated by ultrasound. In a further aspect of the present invention therefore there is provided a sonochemistry apparatus comprising a sonicator device as described above. A yet further aspect of the invention lies in the use of the sonicator device described in performing sonochemistry reactions.

In a further aspect of the invention there is provided a method for determining the presence or otherwise of a particular biological agent in a sample comprising the steps of introducing the sample into a sonicator device as described above, operating the ultrasonic transducer at the resonant frequency for a period of time and looking for the presence of known material in the Iysed sample. For instance if one is screening for a particular bacterium one may want to identify the intracellular DNA associated with that bacterium. Hence by Iysing the sample and looking for the presence or otherwise of the known DNA a screening process for that bacterium is provided. Conveniently the step of looking for the presence of known material in the Iysed sample comprises the step of adding a reagent to the chamber, either before or after sonication, which reacts with known material to give an indication that such known material is present.

Advantageously the reagent is one which reacts with the known material to generate light. Therefore the sample could be added to the chamber and sonicated and the presence or absence of light emission could then be used as an indicator or whether a particular biological agent was present or not.

The invention will now be described by way of example only with respect to the following drawings, of which; Figure 1 shows a micro-sonicator according to one embodiment of the present invention in a) plan view and b) cross section, Figure 2 shows the acoustic output of a sonicator as shown in Figure 1 as a function of driving frequency, Figure 3 shows the impedance of the sonicator as a function of frequency when a) filled with air and b) filled with water, Figure 4 shows the acoustic output of a sonicator and the change over time thereof when filled with water aerated to different amounts, Figure 5 shows a feedback control system for a sonicator, Figure 6 shows a feedback control system for measuring the impedance of the sonicator, Figure 7 shows a circuit diagram of a suitable feedback control system, Figure 8 shows the measured bioluminescence and acoustic output as a function of frequency for Iysis of E.coli (genetically modified), and Figure 9 shows the measured chemiluminescence from Bacillus globigli as a function of the sonication frequency via the luminol reaction.

Figure 1a shows a plan view of a micro-sonicator according to the present invention and Figure 1 b shows the cross-section along the section AB. The sonicator, generally indicated 2, has a Iysis chamber 4 formed in a silicon substrate 6. The chamber 4 is covered by a glass membrane 8 which is approximately 250 microns thick. The diameter of the chamber 4 is approximately 6mm with a depth in the range of 80 to 350 microns, in this instance 200 microns. The silicon substrate has channels etched therein to form an inlet 10 and an outlet 12 to the chamber 4. The channels were in the region of 100 microns wide. The Iysis chamber therefore forms part of a flow through system for sample analysis. On the surface of the glass membrane 8 is a piezoelectric transducer 14 of PZT of diameter 3mm and thickness 200 microns connected to a driving source via electrodes (not shown).

In use a sample is flowed into the chamber 4 using any conventional driving force such as gas over pressure, syringe driver or vacuum. Once the sample is located in the chamber an AC driving voltage is supplied to the piezoelectric transducer 14. This causes the transducer 14 and hence membrane 8 to vibrate, supplying ultrasonic energy to the sample and causing Iysis of the cell or spore sample. The amplitude and frequency of vibration of the piezoelectric transducer 14 are controlled so that the device is driven at the resonant frequency of the chamber 4.

The use of silicon and glass to define the chamber 4 results in a relatively stiff structure with a high quality, or Q. factor. This means that the system has a reasonably well defined resonance frequency at which energy will be efficiently coupled into the system.

Coupling at other frequencies will be relatively poor and the device will operate inefficiently.

The apparatus is therefore different to prior art devices using plastic or other less rigid material which has a lower Q factor and therefor a lesser dependence on resonant frequency. Unlike US6,440,725 which is driven at the resonant frequency of the target cells the present invention operates at the resonant frequency of the chamber.

The resonant frequency of the chamber is a function of the device itself and is dependent upon the physical structure thereof. However the resonant frequency will also depend upon the nature of the material within the chamber. The resonant frequency will depend upon the sample and will also change due to the presence of air bubbles or impurities in the chamber. Cellular or spore debris which are generated as a direct result of sonication will also increase during operation and thereby bring about a change in the resonant frequency over time, indeed the presence and concentration of biological material in the chamber will also effect the resonant frequency of the device. Therefore the resonant frequency can change over time as the sample changes or bubbles pass through the system. This is particularly an issue in a flow through system such as shown in Figure 1.

Physical measurements made as a function of frequency of the device shown in figure 1 show that the structure is strongly resonant. The acoustic output from the device was measured as a function of frequency. A microphone was placed above the sonicator being driven at a frequency f. A spectrum analyser was used to measure the sound level generated at the microphone at frequency f. The driving frequency was swept to generate a response spectrum. Figure 2 shows the result for a sonicator filled with air.

The response is seen to be sharply peaked 20 at a frequency of 54 kHz indicating a strong resonance at this frequency. When the device was filled with water the resonance peak shifted to 109 kHz. The Q factor, i. e. the resonant frequency divided by FWHM, can be seen to be around 30 for this device when filled with air.

An alternative method of measuring the response of the sonicator is to measure the electrical impedance of the sonicator as a function of frequency. Figure 3a shows the results of the case for impedance measured with air in the chamber. A strong peak 22 is observed at 54 kHz which is a similar frequency response to that found in the acoustic measurement. Figure 3b shows the measured impedance when the device is filled with water. Here a frequency peak 24 is observed at 109 kHz which is again similar to the acoustic measurements. This shows that the device has a strong resonance at a resonant frequency and that the particular resonant frequency depends upon the conditions within the Iysis chamber.

When using real samples the effect of bubbles in the sample changes the resonant properties of the device. The acoustic response of a sonicator device as shown in Figure 1 was measured as aerated water was flowed through the device and the results are shown in Figure 4. The resonant frequency was measured at three different times with different bubble configurations within the chamber. It can be seen that the resonant frequency changed from 54 kHz to 52 kHz and then to 46 kHz as the conditions were changed It can also be seen that the resonant peak has a relatively narrow bandwidth and thus at frequencies away from the resonant peak for the prevailing conditions energy is inefficiently coupled into the device. Again the device has a Q factor in the region of 30.

Therefore the present invention includes a feedback control mechanism to ensure that, for efficient Iysis, the device is continually operated at the resonant frequency for the particular conditions. A control system is illustrated schematically in Figure 5. Here the sonicator 2 is monitored by a monitoring means 40. The monitoring means could be an acoustic sensor, such as a microphone, as previously described. However the skilled person will be aware that other means of monitoring the resonant frequency could be used, for instance a vibrometer, such as a laser vibrometer, could be used to measure the movement of the glass membrane at the driving frequency. Alternatively a strain sensor could be incorporated into the structure to measure the movement of the glass.

The light output from a bioluminescent or chemiluminescent reaction which depends upon the amount of Iysed material present could also be used as an indicator as will be more fully described below. Therefore the monitoring means could comprise a photodetector. Similarly in some applications it may be possible to observe the degree of sonoluminescence, i.e. light generated through sonic stimulation, and use the light intensity as a measure of resonance.

The output of the monitoring means 40 is input to a control unit 42 which controls the drive unit 44 to drive the piezoelectric transducer of the sonicator 2 at the resonant frequency. This therefore gives real time performance control and ensures that the energy is always efficiently coupled to the device.

One useful embodiment uses the impedance of the sonicator to determine the resonant frequency of the device. Figure 6 shows a suitable control system which is similar to that shown in Figure 5 and therefore like components have like numerals. The sonicator 2 is driven by the driving unit 44 which is a frequency generator. The frequency generator is driven by control unit 42 which is a suitably programmed processor. The impedance of the sonicator is measured by measuring the voltage across a resistor 46. As will be well known in the art the voltage across the resistor is proportional to the current through it which, for a particular voltage, is inversely proportional to the impedance of the sonicator.

Measuring the resistor voltage therefore allows calculation of the impedance and hence an indication of resonance without requiring any additional sensors.

The voltage across the resistor is measured and envelope detected using an envelope detector before being filtered to remove any remaining ACcomponent. The processor can then measure the DC component. The use of the processor 42 allows a range of tracking routines and algorithms to be applied allowing for very flexible use. The processor could control the frequency generator 44 to perform a linear scan of the frequency range in which resonance is likely to lie. For the system described above a suitable range may be 1 OkHz to 200kHz although this range could be extended if required. A full linear scan could be achieved in around 0.3 seconds. The resonant frequency is that frequency at which the maximum current is demanded by the sonicator, i.e. lowest impedance. The driving frequency can then be set to track the determined resonant frequency, i.e. small regular scans conducted around this determined frequency. Alternatively the driving frequency could be locked until the measured current drops below a set threshold at which time another scan is initiated. The skilled person would be aware of various control schemes which could be applied for particular embodiments.

A suitable circuit diagram for a control means is shown in Figure 7.

In order to ensure that Iysis was optimised at the resonant frequency insitu monitoring of Iysis was performed. In order to monitor Iysis biochemical reagents were introduced into the Iysis chamber which result in the emission of light when a cell is broken open. A strain of Ecoliwas used which has been genetically modified to give a high level of expression for the luciferase protein. A commercially available reagent was added to the Iysis chamber as supplied by Promega_ which is a proprietary formulation containing ATP and luceferin. The cells are suspended in buffered pH 7.4 phosphate buffer to prevent auto cell Iysis. When Iysis occurs the cell breaks open and the luciferase is released which then reacts with ATP and luciferin contained in the supernatant resulting in the emission of light.

The emission of light for E.colicells undergoing Iysis was measured at a range of frequencies using a photodetector. The cells and reagents were flowed into the sonicator chamber and stimulated at a time t at a certain frequency. The light intensity was then measured immediately afterwards. The frequency was then changed and the sample stimulated again. The acoustic output of the cell was also measured using a microphone in a similar manner to that described above. Figure 8 shows the results.

The solid curve 50 represents the acoustic output of the device with frequency. Again a strong resonant peak is observed. The light output can, dashed curve 52, also be seen to have a resonant peak which is closely linked to the resonant frequency as determined by the acoustic output. As the light output is linked to the amount of luciferase undergoing reaction, which is in turn linked to the number of cells undergoing Iysis, it can be seen that Iysis is most efficient around the resonant frequency.

A similar experiment was performed on a spore forming bacterium, B. globigii. Here, a chemiluminescent assay was performed which is sensitive to any haematin containing species such as cytochromes released from within the endospore or its outer coating when the spores are disrupted. The freed haematin takes part in a luminol reaction to give emission of light at 420nm. Figure 9 shows the light output measured during sonication of B.globigli spores at 1 o6 cfu/ml which were sonicated at a frequency between 95 and 117 kHz. Again there is a sharp frequency dependence which correlates with the acoustic output (not shown) from the sonicator.

A chemiluminescent or bioluminescent reaction such as described above could therefore be used as another method of determining the resonant frequency of the Iysis chamber for use in the feedback control loop.

The present invention therefore provides a more efficient sonicator device than the prior art by ensuring that the device operates at the resonant frequency of the device, even in changing conditions, when Iysis is required. It should be noted however that the chamber could have multiple uses and in some situations it may be desired to operate off the resonant frequency. For instance before or during Iysis it may be wished to supply ultrasound to the chamber at a level that does not causes any substantial damage to the cells or other biological material but which mixes the contents of the chamber. Therefore the control means may be adopted to track the resonant frequency when Iysis is required but operate at a different frequency to perform other functions, such as mixing or promoting sonochemical reactions. In this regard it is noted that in some sonochemistry applications it can be advantageous to have two ultrasonic transducers working; simultaneously at different frequencies. The present invention could easily be provided with two or more transducers capable of independent operation and the control means may be adapted to control each transducer either simultaneously or sequentially at the same or different frequencies and either at resonant frequency for Iysis or at some other frequency for another application.

Referring back to figure 1 it can be seen that the device shown therein has inlet and outlet channels 10 and 12 respectively. As mentioned these channels can be formed in the same silicon substrate as the Iysis chamber 4. Indeed the inlet or outlet channels 10 1 and 12 could be connected to other chambers or components in a micro-fluidic system.

An entire micro-fluidic circuit could be formed in a single substrate using micro-machining techniques well known to one skilled in the art of micro-machining. This allows formation of compact micro-fluidic circuits for performing particular assays in a cost effective I manner. Various reagents or detergent based materials could be added to the sample before and/or after the Iysis chamber as required as would be understood by one skilled in the art. It should be noted however that the invention could equally be applied to a non flow through system, i.e. a system without inlet 10 and outlet 12 and operation at the resonant frequency would still give most efficient Iysis.

Some designs of sonicator chamber may permit the device to have other uses in a microfluidic circuit. As shown in figure 1 the inlet and outlet channels are tapered and arranged so that the inlet channel 10 widens nearer the chamber 4 whereas the outlet channel 12 narrows nearer the chamber 4. In one embodiment the narrow part of each channel is approximately 80 microns and the wide part 214 microns with the channels being 1093 microns long. This design does allow for Iysis to occur when the piezoelectric transducer is driven at an appropriate frequency, i.e. the resonant frequency. The design is similar however to a pump device described in US patent application 6,203,291. Here it is noted that using a tapered nozzle design as described and vibrating a membrane at a frequency of approximately 200 Hz water can be pumped through the system in a preferential direction without needing valves.

It is possible therefore to operate the embodiment of the invention having such a tapered nozzle design in two modes. The piezoelectric transducer could be driven at a low frequency, say less than 2kHz in order to pump material through a flow through system.

This pumping frequency is far too low to cause Iysis or disrupt the material at all - hence its usefulness for pumping. Once a sample is located in the chamber 4 though the frequency of excitation could be changed to that of the resonant frequency - in the range -150 kHz say. Excitation at this frequency as mentioned can cause efficient Iysis.

Other designs of chamber are possible though as will be apparent to the skilled person.

The exact mechanism by which Iysis occurs is not fully understood but it is believed that cavitation may play an important role. Cavitation is the formation of small very high energy bubbles when high energy density ultrasound is applied. There are optimum frequencies for cavitation these are typically in the range 1 OkHz to 1 MHz. Thus the optimum sonicator will be designed to have its resonance in this range. The input and output channels being deigned as nozzles may also play a part because of the high shear forces experienced in the vicinity of these nozzles.

Cavitation may be optimised when the sample actually has dissolved gas in it. This could come out of solution on sonication and give rise to bubbles which would shift the resonant frequency. Therefore the mechanism by which Iysis occurs could also change the parameters of the cell - however the resonant frequency tracking of the present invention allows efficient real time operation.

Large bubbles can form in the sonicator and these are a nuisance as effectively they reduce the volume of material being treated. They can also change the effective Q factor and shift the resonant frequency. This effect may be reduced by operating the devices so that the output channel is arranged to be is vertical. In this way the bubbles 'float' to the top and flow out of the system.

Although silicon has been described herein as the substrate material other semiconductor materials could be appropriate or any other material susceptible to micro- machining. Indeed micro-machining techniques could be used to produce a master for embossing the required structure into a suitable thermoplastic material. As mentioned however it is preferable that the material is relatively stiff so that the system has a high Q factor. The sonicator device itself is therefore preferably attached to a solid support to provide further stiffness and ensure that only the membrane is moved by the piezoelectric transducer. Similarly although glass is a convenient material for the membrane 8 other flexible materials, again preferably with a high Q factor could be used.

The membrane could be made from silicon as well - in which case a higher Q might be possible Further the Iysis chamber could be provided with other features to either aid efficient Iysis or subsequent assays or both. For instance particles could be added into the Iysis chamber, for example glass beads with a diameter of say 50 microns. The glass beads could increase the surface area to volume ratio within the chamber. This could result in a greater amount of cavitation as energy is supplied to the sample which could aid Iysis.

Obviously the addition of glass beads will change the resonant frequency of the device but the present invention as described above will still automatically find the resonant frequency and operate at that frequency. For a similar reason a chamber could be designed with a number of protrusions within the chamber.

Also it is possible that the high shear forces in the vicinity of the inlet and outlet nozzles and 12 could aid Iysis. The chamber could be provide with additional nozzles or channels to increase the shear forces within the chamber.

Claims (34)

1. A sonicator device comprising a chamber formed, at least in part, by a flexible membrane and having an ultrasonic driving means mounted on said membrane characterized in further comprising a monitoring means, for monitoring the performance of the sonicator device and a control unit, responsive to said monitoring means, for allowing, in use, the ultrasonic driving means to operate at the resonant frequency of the device.

2. A sonicator device as claimed in claim 1 wherein the chamber has at least one inlet channel.

3. A sonicator device as claimed in claim 1 or claim 2 wherein the chamber has at least one outlet channel.

4. A sonicator device as claimed in any preceding claim wherein the chamber is formed, at least in part, from a semiconductor material.

5. A sonicator device as claimed in claim 4 wherein the chamber is formed as a cavity in a substrate of semiconductor material.

6. A sonicator device as claimed in claim 4 or claim 5 wherein the semiconductor material is silicon.

7. A sonicator device as claimed in any preceding claim wherein the membrane comprises glass.

8. A sonicator as claimed in any preceding claim wherein the device has a high Q factor.

9. A sonicator as claimed in claim 8 wherein the Q factor with is greater than 15, 20, 25, 30, 50 or 100.

10. A sonicator device as claimed in any preceding claim wherein the ultrasonic driving means comprises a layer of piezoelectric transducer.

11. A sonicator device as claimed in any preceding claim wherein the monitoring means comprises an acoustic sensor.

12. A sonicator device as claimed in claim 11 wherein the acoustic sensor comprises a microphone located adjacent the chamber.

13. A sonicator device as claimed in any of claims 1 - 10 wherein the monitoring means comprises a means responsive to the impedance of the sonicator device.

14. A sonicator device as claimed in claim 13 wherein the means responsive to the impedance of the sonicator device comprises a means for measuring the current through a resistor connected to the sonicator device.

15. A sonicator device as claimed in claim 13 wherein the means responsive to the impedance of the sonicator device comprises a means for measuring the voltage across a resistor connected to the sonicator device.

16. A sonicator device as claimed in any of claims 1 - 10 wherein the monitoring means comprises a means for measuring the vibration of the membrane.

17. A sonicator device as claimed in any of claims 1 - 10 wherein the monitoring means comprises a means for directly monitoring the extent of Iysis within the chamber.

18. A sonicator device as claimed in any preceding claim wherein the control means comprises a processor.

19. A sonicator device as claimed in any preceding claim wherein the control means is adapted to periodically scan the ultrasonic driving means across a frequency range and determine the resonant frequency from the output of the monitoring means.

20. A sonicator device as claimed in any preceding claim wherein the control means is adapted to determine the resonant frequency and the output of the monitoring means at that frequency and initiate a frequency scan when the output from the monitoring means changes by a set amount.

21. A sonicator device as claimed in any preceding claim wherein the volume of the chamber is less than 10 microlitres, less than 5 microlitres or less than 1 microlitre.

22. A method of sonicating a sample comprising the steps of; i) introducing a sample to a chamber comprising, at least in part a flexible membrane connected to an ultrasonic driving means, ii) determining the resonant frequency of the chamber, iii) driving the ultrasonic driving means at the determined resonant frequency.

23. A method as claimed in claim 22 wherein the step of determining the resonant frequency of the chamber comprises the steps of driving the ultrasonic driving means across a range of frequencies and monitoring a characteristic of the chamber which has a turning point at the resonant frequency.

24. A method as claimed in claim 23 wherein the characteristic measured is acoustic output from the chamber.

25. A method according to claim 24 wherein the step of monitoring the acoustic output comprises providing an acoustic sensor adjacent the chamber.

26. A method according to claim 23 wherein the characteristic measured is related to the impedance of the ultrasonic driving means.

27. A method according to claim 26 wherein the characteristic measured is the current through a resistor connected to the ultrasonic driving means.

28. A method according to claim 26 wherein the characteristic measured is the current through a resistor connected to the ultrasonic driving means.

29. A control apparatus for a sonicator device comprising a monitoring means for monitoring the performance of the sonicator device and a control unit, responsive to said monitoring means, for ensuring that, in use, the sonicator device operates at its resonant frequency.

30. A sonochemistry apparatus comprising a sonicator device as claimed in any of claims 1 - 21.

31. The use of a sonicator device as claimed in 1 - 21 in performing a sonochemistry reaction.

32. A method for determining the presence or otherwise of a particular biological agent in a sample comprising the steps of introducing the sample into a sonicator device as claimed in any of claims 1 - 21, operating the ultrasonic transducer at the resonant frequency for a period of time and looking for the presence of known material in the Iysed sample.

33. A method as claimed in claim 32 wherein the step of looking for the presence of known material in the Iysed sample comprises the step of adding a reagent to the chamber which reacts with known material to give an indication that such known material is present.

34. A method as claimed in claim 33 wherein the reagent is one which reacts with the known material to generate light.