WO2008104916A2

WO2008104916A2 - A cell lysis and/or mixing device

Info

Publication number: WO2008104916A2
Application number: PCT/IB2008/050656
Authority: WO
Inventors: Lucas Johannes Anna Maria Beckers; Sergei Shulepov; Maarten A.F. Vervoort
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2007-02-27
Filing date: 2008-02-22
Publication date: 2008-09-04
Also published as: WO2008104916A3

Abstract

The present invention is related to a cell lysis and/or mixing device, comprising a chamber (11) having a volume, wherein said chamber is filled with microparticles (12), and said chamber is adapted to accommodate cells. The device further comprises means (15) to put said chamber under vibrations. Furthermore, it is provided that the microparticles within the chamber assume between 20 and 75 % of the chamber's volume (Fig. 4). The present invention further provides a process for lysing and/or mixing cells, wherein cells are put in a chamber which is filled with microparticles, the chamber is set under vibrations, wherein further the microparticles assume between 20 and 75 % of the chamber's volume.

Description

A CELL LYSIS AND/OR MIXING DEVICE

FIELD OF THE INVENTION

The present invention relates to a cell lysis and/or mixing device which can be used in a microfluidic device environment.

BACKGROUND OF THE INVENTION

The biotechnology sector has directed substantial effort toward developing miniaturized microfluidic devices, often termed labs-on-a-chip (LOC) or micro total analyses systems (microTAS), for sample manipulation and analysis. These systems are used for detection and analyses of specific bio-molecules, such as DNA and proteins, and are for example known from previous patent applications by the applicant. In general micro-system devices contain fluidic, electrical and mechanical functions, comprising pumps, valves, mixers, heaters, and sensors such as optical -, magnetic - and/or electrical sensors. A typical molecular diagnostics assay includes process steps such as cell lysis, washing, amplification by PCR, and/or detection.

The term "microfluidic device" as used herein relates to an apparatus for molecular diagnostic applications, such as a "lab-on-a-chip" ("LOC") or a micro total analyses system (mTAS). Recently, the biotechnology sector has directed substantial effort toward developing microfluidic devices, for manipulation and analysis of biological samples. These systems are used for detection and analyses of specific biomolecules, such as nucleic acids and proteins. These devices are highly integrated devices with a miniaturized structure, and being prepared for high throughput procedures. Due to the high degree of miniaturization, they are based on microfluidic principles, enabling only small sample volumes. A typical LOC for nucleic acid analysis comprises a sample reception chamber, a cell lysis device, a PCR device and a sample analysis device, all integrated on a small chip. The term "cells" as used herein relates to biological cells, i.e. prokaryotic and eukaryotic cells, as well as to viruses, although the latter do not fall under the biological definition of the term "cell". Prokaryotic cells include gram negative and gram positive bacteria, as well as algae, whereas Eukaryotic cells include plant cells, fungal cells and animal cells. The latter include, among others, mammal cells, particularly human cells.

All cells in the above meaning - despite viruses, which have a protein capsid - have a plasma membrane, i.e. protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Moreover, plant cells, prokaryotic cells, algae and fungus cells have a cell wall which provides physical support.

Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of β-glucan. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. In order to analyse cell contents, as for example the genome (DNA contents), the proteome (protein contents), the methylom (DNA methylation patterns) or the transcriptome (RNA contents), as well as intracellular structures, like organells, the cytoskeleton and so forth, the cells have to be disrupted, i.e. their capsid, cell membranes and/or cell walls have to be disrupted. The term "cell lysis" as used herein refers to such disruption of cells in order to provide their content for further analysis, modification, or use. It is easy to understand that the more rigid the outer cell structures, i.e. capsid, cell membranes and/or cell walls are, the more difficult it is to disrupt them.

BACKGROUND ART

Generally, there are two ways to disrupt a cell in order to lyse it, namely the application of physical force or a chemical agent. The different varieties will be discussed in the following:

Hypotonic shock Quite frequently, simply lowering the ionic strength of the media will cause the cells to swell and burst. In some cases it is also desirable to add a mild surfactant and some mild mechanical agitation or sonication to completely disassociate the cellular components. Due to the cost and relative effort to grow these cells, there is often only a small quantity of cells to be processed, and preferred methods for cell disruption tend to be a manual mechanical homogenizer, nitrogen burst methods, or ultrasound with a small probe. For cells that are more difficult to disrupt, such as bacteria, yeast, and algae, hypotonic shock alone generally is insufficient to open the cell and stronger methods must be used. These organisms have cell walls that must be broken to allow access to intracellular components. These stronger methods are discussed below. Freeze/Thaw

The freeze/thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols. Chemical agents Quite often, chemical agents are being used for cell lysis, like phenol, chloroform and/or isoamyl alcohol. These agents have in common that they are quite toxic. Moreover, due to their volatility they are difficult to use in a microfluidic environment, as the low volumes which can are to be used in this environment tend to evaporate. Other agents comprise strong alkalines, like NaOH, KOH, LiOH and the like. This kind of lysis is often termed "alkalic lysis" Enzymes/Chemical agents

The use of enzymatic methods to remove cell walls is well established for preparing cells for disruption or for preparation of protoplasts (cells without cell walls) for other uses such as introducing cloned DNA or subcellular organelle isolation. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources (e.g. the snail gut enzyme β-glucuronidase for yeast, or lysozyme from hen egg white for prokaryotes). A similar approach makes use of chemical agents like phenol. Both methods have in common that the cell lysis result is somehow polluted with the respective agent, which may have a negative effect on the succeeding analysis.

Sonication Another method for cell disruption applies ultrasound (typically 20-50

KHz) to the sample (sonication). In principle, the high-frequency is generated electronically and the mechanical energy is transmitted to the sample via a metal probe ("sonotrode") that oscillates with high frequency. The probe is placed into the cell- containing sample and the high-frequency oscillation causes a localized high pressure region resulting in cavitation and impaction, ultimately breaking open the cells

Cell Bomb

Yet another method for cell disruption is rapid decompression, often termed the "cell bomb" method. In this process, cells in question are placed under high pressure (usually nitrogen or another inert gas up to about 25,000 psi) and the pressure is rapidly released. The rapid pressure drop causes the dissolved gas to be released as bubbles that ultimately lyse the cell.

High-shear mechanical methods.

High-shear mechanical methods for cell disruption fall into three major classes: rotor-stator disrupters, valve-type processors, and fixed-geometry processors. These processors all work by placing the bulk aqueous media under shear forces that literally pull the cells apart. These systems are especially useful for larger scale laboratory experiments (over 20 mL) and offer the option for large-scale production.

Other methods

Other methods sometimes used include NaOH-SDS solubilization, French press treatment, Laser Microbeam-Induced Cell Lysis or mutanolysin treatment. Despite the disadvantages already discussed, all the above methods have in common that they are difficult to be integrated in a microfluidic device, such as a lab on a chip.

Agitated Beads

Yet another method for cell lysis makes used of agitated particles, like glass or ceramic beads. In this approach, lysis is usually achieved through mechanical disruption of cells caused by shear forces and/or impact forces exerted to the cells by the agitated beads.

Such a device is for example disclosed in US 6887693. However, while this approach is well established for normal sized laboratory applications, it is not easily transferred to micro fluidic device environments. Due to the fact that the sizes of the particles have to be decreased when transferring the method to microdevices, the latter tend to be small of weight, which results in low kinetic energies and thus small shear or impact forces, even if agitated to a high degree, and thus poor lysis results, particularly for rigid cells. This means that a mere downscaling of the bead based cell lysis devices known from the state of the art does not work in micro fluidic device environments. Another drawback of the above mentioned method is that they are not suitable for use in a dissposable environment. However, in order to accelerate high throughput cell diagnosis and analysis, and to make these processes more const efficient, a disposable concept is crucial.

OBJECT OF THE INVENTION

It is therefore the object of the present invention to provide a device for cell lysis and/or mixing which can easily be integrated into a microfluidic device environment, and which is suitable to disrupt or lyse even cells with rigid cells membranes or cells walls, like gram-poitive bacteria, algae, plant cells or yeast, while avoiding the above mentioned disadvantages.

This object is met with a device according to the independent claims. The dependent claims provide preferred embodiments.

SUMMARY OF THE INVENTION

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

According to the invention a cell lysis and/or mixing device is provided, which comprises a chamber having a volume, wherein said chamber is filled with microparticles, and said chamber is adapted to accommodate cells. The device further comprises means to put said chamber under vibrations. Furthermore, the microparticles within the chamber assume between 20 and 75 % of the chamber's volume.

In the following the term "microparticles" will be used synonymously for the term "beads". Likewise, the chamber will as well be termed "bead bed reactor". Typically, bead lysing devices known from the state of the art have a filling ratio of 25 % or less. By using the specified filling ratio according to the invention the frequency of bead collisions is increased, which equalizes the decrease in kinetic energy due to smaller particle sizes, and thus results in a very effective cell lysis.

As set forth above, in such a device, the lysis of the cells relies mainly on the influence of shear forces and impact forces exerted by the microparticles to the cells.

The first mechanism (shear forces) depends mainly on the cell Weber number:

We = pV_r ² _elD_p /2a (eq. 1)

Where p is the fluid density,

is the relative velocity between the beads, Dp is the cell diameter and σ is the cell surface tension. When We > 6 cell disruption takes place. For a typical Gram positive bacterium this happens for a relative velocity of about 5-10 m/s.

The second mechanism (impact forces) relies on the principle that, when a cell is trapped between two beads during their collision, it is, as a consequence, squeezed to break. Cell rupture takes place here when the kinetic energy of the colliding beads exceeds the elastic energy stored in the cell. Typically, 60-80 % of volumetric deformation, which corresponds to more than 2 % of the surface deformation of lipid layers, is required to break the bacteria. Much lower bead speeds are needed in this case

(order of 0.3 m/s) as compared to the first mechanism. Inter-beads collisions are therefore the preferred mechanism of cell rupture during lysis. This rupture mechanism is efficient only if a minimum collision velocity is achieved, if the collision frequency is high and if the beads are well mixed in the solution.

The consequence is that to enhance the number of collisions without increasing too much the viscosity of the suspension, a value of volume fraction between 30 and 50% should be achieved. The collision Stokes number is defined here as: St_c = p_pV_relD_p /9μ (eq. 2)

This number is a measure of the inertia of the bead as opposed to the viscous force exerted on the bead by the fluid. If the Stokes number is low (less than about 40), then the collision will be severely damped by viscosity and no significant exchange of kinetic energy will take place between the beads. For Stokes numbers larger than about 40, a partially elastic collision will take place. In order to have effective collisions during the lysis process a Stokes number at least larger than about 40 is needed. The Stokes number for glass beads of 500 μm diameter immersed in water and colliding with relative velocity of about lm/s is for example about St_c~l70. As becomes clear from these considerations, it is, in order to promote cell lysis in a miniaturized bead lysis device, beneficial to promote the development of impact forces rather than of shear forces inside the bead bed reactor.

This goal is achieved, among others, when the chamber is filled up to the claimed filling ratio, as thereby a "volume deformation behavior" of the bead bed reactor is promoted (see Fig. 3), rather than a "volume displacement behavior". Through this achievement, vertical oscillations of the microparticles/beads are promoted within the reactor rather than lateral or circular movements of the beads, and thus impact forces exerted to the cells are increased despite the use of small particle sizes. However, a "sand bag" behavior of the chamber, which occurs in case the filling ratio is too high (i.e., above the specified filling ratio) due to increased viscosity, and for which the chance of bead collision - and thus effective cells lysis - decreases, is avoided within the specified filling ratio.

In a preferred embodiment, the filling ratio is in the range of 30 and 50%. Applicants have found that, when choosing such filling ratios, even rigid cells can efficiently be lysed within short. The term "cells" as used herein relates to biological cells, i.e. prokaryotic and eukaryotic calls, as well as to viruses, although the latter do not fall under the biological definition of the term "cell". Prokaryotic cells include archaebacteria, gram negative and gram positive bacteria, as well as algae, whereas Eukaryotic cells include plant cells, fungal cells and animal cells. The latter include, among others, mammal cells, particularly human cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In a preferred embodiment, the chamber/bead bed reactor has a roughly circular cross section. In this context, the term "roughly circular" relates to ovoidal cross sections, ellipsoidal cross sections, polygonal cross sections with rounded edges or circular cross sections. More preferred, the chamber/bead bed reactor has a cylindrical shape, based on the above mentioned cross sections. The chamber/bead bed reactor may have a diameter of between 2 and 30 mm, and a height of between 0,5 and 10 mm. In as preferred embodiment, the diameter ranges between 5 and 15 mm, and the height ranges between 2 and 5 mm.

The chamber/bead bed reactor may, in a preferred embodiment, consist of a bottom part ("lysis bed") and a top part ("lid", see Fig. 7). Preferred chamber/bead bed reactor materials comprise thermoplastics, particularly thermoplastic elastomers. Other preferred materials comprise guttapercha, latex, and rubber. In yet another preferred embodiment, the chamber/bead bed reactor or parts thereof may be adapted to be produced with injection molding techniques.

In another preferred embodiment, the microparticles/beads have a roughly circular shape. In this context, the term "roughly circular" relates to ovoidal shapes, ellipsoidal shapes, polygonal shapes with rounded edges, circular shapes or spherical shapes.

The beads consist, in preferred embodiments, of glass, ceramic or a metal. In a preferred embodiment, the beads may consist of a magnetizable material, like stainless steel. In this embodiment, the vibration of the beads may further be supported by application of an AC electromagnetic field. The device according to the invention may, in a preferred embodiment, be characterized in that the chamber comprises at least one inlet and one outlet. In this embodiment, a flow through chamber may be accomplished, in which suspended cells can be pumped into the chamber through an inlet, lysed and/or mixed with other agents therein, and then be pumped out of the chamber through the outlet, in order to forward the lysed or mixed contents to other devices on a LOC environment.

In a preferred embodiment, the inlet and/or the outlet comprises a sieve or a filter. These sieves or filters serve to keep the particles, particularly the beads, within the chamber, while letting cells pass through. These sieves or filters may consist of dedicated filter cartridges introduced in the chamber/bead bed reactor in the region of the inlet and/or outlet, e.g. by clamping. However, these filters have to be fixed or immobilized in the chamber individually, which causes additional hassle and effort and increases the manufacturing costs of such a chamber/bead bed reactor, and makes it thus unsuitable for use in a disposable context. For this reason, it is preferred that the filters or sieves are accomplished by creating pores right in the chamber/bead bed reactor walls in the region of the inlet and/or outlet. This can for example be accomplished by laser drilling right into the wall material of the chamber, with which pores can be created which a have diameter of down to 90 μm. In this embodiment, an additional mixing effect can be achieved, as by forcing liquid through such a sieve, a spraying effect is supported which leads to a better distribution in the chamber of the liquid containing the cells to be lysed. This means that in this embodiment, the laser drilled sieves doubleact as filter and mixing devices, while they are easily implemented into the chamber at low manufacturing costs, which makes this embodiment suitable for use in a disposable context.

The inlet may comprise at least one additional duct for chemical and/or biological agents. These agents may for example be selected from the group consisting of cell lysis agents, cell lysis enzymes, nucleic acid amplification agents comprising nucleotides, polymerases, primers, and the like. This embodiment draws large benefit from the fact that the cell lysis device according to the invention is also a highly efficient mixing device. In a preferred embodiment, the chamber may comprise an elastic bottom part and an elastic top part. In this embodiment, elastomer materials can be used, like thermoplastic elastomers, latex, rubber, guttapercha and the like. This embodiment, together with the high filling ratio of the chamber, promotes the volume deformation behavior of the chamber/bead bed reactor, and thus increases the impact forces exerted on the cells

An example for a preferred thermoplastic elastomer is "Santoprene", which has the following properties that make it suitable for the application according to the invention: - Tensile stress at 100% (23 ⁰C) 2.1 MPa

Tensile stress at break (23 ⁰C) 5.2 MPa Tensile strain at break (23 ⁰C) 400%

With these data, the skilled person will readily be able to select other suitable chamber materials, simply from material data sheets, encyclopediae, material databases and the like, without involving an inventive step.

In a preferred variant of this embodiment, the side walls of the chamber can consist of the same material, while having a larger thickness than the bottom part and the top part. This results in smaller elasticity of the side walls as compared with the top and bottom part, which again supports the above mentioned "volume deformation behavior" of the chamber/bead bed reactor (see Fig. 3).

On the basis of the above mentioned cell sizes, preferred bead sizes and pore sizes of the filters or sieves can be determined. In a preferred embodiment, the bead have thus sizes between 10 and 1000 μm. More preferred, bead sizes range between 100 and 500 μm. Even more preferred, bead sizes range between 300 and 400 μm. As pores sizes of the filters or sieves should be selected to let cells pass, but keep the particles in the chamber/bead bed reactor, pore size diameters may vary between 5 and 800 μm, depending on the beads used. In general, they should be about 50 % smaller than the beads used.

Furthermore, the device may, in a preferred embodiment, comprise a stamp which sets the chamber under vibrations. This stamp, or piston, may for example be disposed above or below the chamber. It may be as well be coherent (or non- coherent) to the chamber's top or bottom. In other embodiments, a dedicated stinger may be used to transmit the vibrations from the stamp to the chamber.

In a preferred embodiment, the vibrations imposed on the chamber/bead bed reactor have a frequency between 2 and 1000 Hz. More preferred, the vibrations are in the range of 10 to 300 Hz.

The amplitude of the vibrations lies, in a preferred embodiment, in a range of between 5 and 200 % of the chamber's height. In a preferred embodiment, the amplitude lies in a range of 20 and 75 %. This means, for example, that with a given chamber height of 3 mm, the vibration amplitude should be in the range of 0,75 and 2,25 mm.

The stamp or piston itself may be set under vibrations by means of a pneumatic device. Other possibilities comprise that the stamp is spring-loaded, and set under vibrations by means of a rotating excenter, or with help of an electromagnetic coil, i.e. similar to a conventional loudspeaker. Another option is a piezoelectric transducer being coupled to the chamber. However, the skilled person will readily select other methods to drive the recited stamp or piston in order to achieve the above mentioned frequency and amplitude values.

Furthermore, a microfluidic device for analysis of a fluid cell sample, especially for molecular diagnostics applications is provided, wherein this device comprises a substrate having a surface with at least one micro channel structure thereon; at least one detecting, controlling and/or processing element; at least one reception chamber for receiving the fluid sample, wherein the reception chamber is fluently connected with at least one micro channel; and a cell lysis and/or mixing device according to any of the aforementioned claims. Such a device falls under the definition of the above introduced "lab-on-a- chip" ("LOC") or micro total analyses system (mTAS). With such device, very small biological samples containing cells can by lysed and analysed.

The microfluidic device can be designed in such way that a number of same or different fluid sample processing, detecting and/or controlling steps can be carried out separate, simultaneous and/or subsequent thereon. As used herein, the term "detection means" or "detecting element" refers to any means, structure or configuration, which allows one to interrogate a fluid sample within the sample-processing compartment using analytical detection techniques well known in the art. Thus, a detection means may include one or more apertures, elongated apertures or grooves which communicate with the sample processing compartment and may allow an external detection apparatus or device to be interfaced with the sample processing compartment to detect a fluid sample, also referred to as analyte, passing through the microfluidic device.

Preferred examples for such detection means are microassays for proteins and/or nucleic acids, which comprise antibodies (immunoassays) and/or nucelic acid probes (hybridization assays). These devices are commonly known under the term "biochip".

Moreover, the device may contain means for nucleic acid amplification, like a PCR device or a device for isothermal amplification, like nucleic acid sequence base amplification (NASBA). In these embodiments, the device comprises a heating/cooling means, like a peltier element. However, the skilled person may select from his knowledge any suitable heating/cooling means.

In another preferred embodiment, the bead bed reactor may by itself serve as an amplification device for isothermal amplification of RNA, like the above identified NASBA. In this embodiment, the beads may doubleact as means for RNA precipitation. The beads may comprise special surface modifications in order to promote RNA binding and/or precipitation.

Furthermore it can be provided that a high salt solution may be used in order to precipitate (floculate) proteins and RNA/DNA molecules onto the beads. In the case of a bead bed reator with a high filling ratio, as claimed herin, a high surface area is present for this floculation. Other materials released from the cells may then be washed out, and thereafter proteins and RNA/DNA can be eluted with use of a suitable buffer. This means that, in this embodiment, the bead bed reactor may as well doubleact as a protein and RNA/DNA purification device. The term "fluid cell sample" is used to refer to any compound or composition containing cells, preferably in suspension, which can be pumped through the micro channel system.

The microfluidic device may furthermore comprise at least one membrane, wherein the membrane covers the upper surface of at least one micro channel structure arranged on said substrate leakage proof, and whereby movement of said membrane causes a pump action on fluid located in said reception chamber in said micro channel and/or causes a valve action on fluid directed through said micro channel; at least one device for actuating the movement of the membrane, comprising pressure and/or vacuum generating means; wherein the reception chamber is formable between the membrane and the substrate.

The term "membrane" as used herein refers to a foil which is impermeable to at least water based liquids. This means that no diffusion will take place through this type of membrane. The size of the membrane may be selected so that the membrane completely or partly covers the upper surface of the substrate. It is most preferred that the membrane covers the micro channel system. An up and down movement of said membrane causes a pump action or valve action so that fluid located in said micro channel system is transported or stopped in the micro channel system. An up movement of the membrane causes a suction function and a down movement of the membrane forces a fluid sample flow and/or causes a valve function. In order to apply pressure and/or vacuum to the membrane, the membrane is in contact with pressure and/or vacuum means. Pressure means comprising gas pressure and/or mechanical pressure means such as plungers or there like. The pressure and vacuum means are not in contact with the fluid sample since the membrane has a fluid sealing function. The pressure and/or vacuum means actuate the upper surface of the membrane at specific areas so that defined areas of the membrane can be lifted up and down only. It is preferred that the predominant part of the membrane surface is fixed by means of a support plate, also referred to as fixture. The support plate can comprise at least one recess, hole or conduit so that the membrane can be moved up and down. Furthers, a recess of the support plate having no hole or conduit can function to receive a membrane up-movement caused by fluid sample flow. Vacuum and/or pressure means can be operative connected to at least one recess, hole and/or conduit of the support plate to actuate the pump and/or valve function of the membrane. The membrane area having a valve and/or pump function is arranged adjacent and/or above the micro channel so that fluid sample in said micro channel can be forced through. It can be preferred that the micro channel adjacent and/or below the movable membrane areas have an enlarged structure, i.e. the channel design at this places has a chamber, compartment or lake-like form.

In case of using plungers it is preferred that the lower surface size of the plunger/s corresponds with the shape of the micro channel, or with the shape of a reception chamber, so that a down movement of the plunger contacting the membrane causes a fluid pressure and/or valve action of the membrane. The plunger can be connected with the upper surface of the membrane, the plunger can be part of the membrane, and/or the plunger fits so in a hole, recess or conduit, that a up and down movement of the plunger actuate the pump and/or valve action of the membrane. If the plunger is part of the membrane, the plunger can be hollow so that a squeezing cause a pump and/or valve action. Thus, the membrane can have a flexible plane shape or a flexible pre shaped design. A membrane with a pre shape design is a membrane that forms at least one compartment or chamber, preferably at least two compartments and chambers. The compartments and/or chambers of the flexible plane membrane

(formed due pump/valve function) and/or of the pre-shaped membrane for receiving fluid sample may have a volume of 0.1 to 100 mm³, preferably 0.5 to 25 mm³ and more preferably 1 to 5 mm³.

Due to the pump and/or valve effect of the membrane at defined areas, i.e. at areas where the membrane is not fixed in its position, fluid sample can be transported through a micro channel system or branched channel system to a desired area. Thus, a fluid sample can be transported out of a reception chamber to a number of different places to be detected, controlled and/or processed. Therefore, the pump system of the present invention may allow a multiple forward and backward fluid transport. Further, the integrated membrane with pump and valve functions provides a fast fluid transport, a small pump and valve dead volume as well as a low vertical range of manufacture. The small dead volume is one benefit of the microfluidic device according to the present invention. In the present invention the total volume of all the micro channels can be preferably less than 1 vol.-%, preferably less than 0.5 vol.-% and more preferably less than 0.1 vol.-% of the total fluid volume. However, it is possible to reduce dead volumes further by pumping air trough the micro channel at the end of the pumping cycle.

The membrane as used according to the present invention is preferably liquid tight, so that liquid fluid does not penetrate the membrane during operation. It may be preferred that the membrane is flexible and/or elastic. Suitable membrane materials are polymers, preferably natural or synthetic rubbers.

To obtain a good pump and/or valve effect of the membrane it may be preferred that the membrane has a thickness of 1 μm to 1000 μm, preferably 25 μm to 500 μm and more preferably 50 μm to 200 μm. If the membrane is to thin there is a danger of deterioration of the membrane, which may result in leakage of the fluid sample. However, if the membrane is to thick, there is a danger of malfunction of the pump and /or valve effect of said membrane with respect to fluid transportation. Most preferred is a rubber membrane having a thickness between 50 μm and 200 μm.

According to the present invention, a substrate surface is at least partly covered with a polymeric layer. The micro channel structure can be formed in said polymer layer by general known techniques. For example, micro channels can be formed by use of laser ablation techniques. A laser ablation process can be used, because it avoids problems encountered with micro lithographic isotropic etching techniques which may undercut masking during etching, giving rise to asymmetrical structures having curved side walls and flat bottoms. The use of laser-ablation processes to form microstructures in substrates such as polymers increases simplicity of fabrication, thus lowers manufacturing costs. Further, microfluidic devices according to the present invention in low-cost polymer substrates have the benefit to be disposable.

In general, any substrate which is UV absorbing provides a suitable substrate in which one may laser ablate features. Accordingly, microstructures of selected configurations can be formed by imaging a lithographic mask onto a suitable substrate, such as a polymer or ceramic material, and then laser ablating the substrate with laser light in areas that are unprotected by the lithographic mask. EP-Al 0 708 331 is directed to laser ablation techniques and is incorporated by reference herein. However, micro channel can also be formed by etching and micromachining techniques used to form systems in silicon or silicon dioxide materials. The microfluidic device can moreover comprise at least one micro channel. Preferably, the microfluidic device comprises a plurality of micro channels, also referred to as micro channel array, formed on a substrate material.

The micro channel structure formed on the substrate can comprises areas where the fluid sample is treated, such as heated, cooled, controlled, reacted, measured and/or analyzed. Further, the micro channel structure comprises areas of pump and/or valve function.

The micro channel can have the form of a channel. However, at places where the fluid sample is subjected to pump or valve effect or treated, such as heated, cooled, controlled, reacted, measured and/or analyzed, the micro channel may have a wider structure, such as a chamber, compartment or lake-like structure.

The substrate material can be selected from the group comprising glass, ceramic, silicon and/or polymer.

The depths of the micro channels may in the range of 5 μm to 200 μm, preferably of 10 μm to 100 μm, further preferred of 20 μm to 50 μm and more preferred 30 μm.

The width of the micro channels at there top opening may in the range of 0.1 μm to 1000 μm, preferably of 1 μm to 500 μm, further preferred of 5 μm to 250 μm and more preferred 100 μm.

Furthermore, the use of a device according to any of the aforementioned claims for cell lysis and/or mixing is provided, as well as a process for lysing and/or mixing cells, wherein a device according to any of the aforementioned claims is used. In another variant, a process for lysing and/or mixing cells is provided, wherein cells are put in a chamber, and the chamber is set under vibrations, wherein further the chamber is filled with microparticles, and the particles assume between 20 and 75 % of the chamber's volume. Furthermore, the use of said cell lysis and/or mixing device, and or said microfluidic device according to any of the aformentioned claims for the following purposes is provided: a) chemical, diagnostic, medical and/or biological analysis, comprising assays of biological samples such as urine, blood, serum, smear probes, biopsies, plasma, forensic samples, sperm smaples, punctations, crusta phlogistica, sputum, b) environmental analysis, comprising analysis of water, dissolved soil extracts, animal faeces, dissolved plant extracts; c) reaction solutions, dispersions and/or formulation analysis, comprising analysis in chemical production, in particular dye solutions or reaction solutions; d) and/or quality safeguarding analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, the figures and the following description of the respective figure and examples, which, in an exemplary fashion, show preferred embodiments of the cell lysis and/or mixing device, or the microfluidic device according to the invention. However, these drawings should by no means be understood as to limit the scope of the invention.

Fig. 1 shows a cell lysis and/or mixing device 10 according to the invention, which can be integrated in a Lab-on-a-Chip environment. The device comprises a chamber 11, which is filled with microparticles 12. The chamber is adapted to accommodate cells, and to be put under vibrations. The particles within the chamber assume between 20 and 75 % of the chamber's volume. The device further comprises an inlet 13a and an outlet 13b, and is thus a flow, in which suspended cells can be pumped into the chamber 11 through the inlet 13a, treated therein according to the invention, and then be pumped out of the chamber through the outlet 13b. Both the inlet 13a and the outlet 13b comprises a sieve 16a, b. These sieves or filters serve to keep the particles, particularly the beads, within the chamber, while letting cells pass through.

Furthermore, the chamber 11 comprises an elastic bottom part 14a and an elastic top part 14b, and a stamp 15 which sets the chamber 11 under vibrations. For this purpose, the stamp 15 is set under vibrations by means of a pneumatic device (not shown). Other possibilities comprise that the stamp is spring-loaded, and set under vibrations by means of a rotating excenter.

The vibrations imposed on the chamber/bead bed reactor have a frequency between 2 and 1000 Hz. The amplitude of the vibrations lies in a range of between 5 and 200 % of the chamber's height. This means, for example, that with a given height of 3 mm, the vibration amplitude should be in the range of 0,75 and 2,25 mm.

Fig. 2 shows the cell lysis and/or mixing device in two different stages of operation. In Fig. 2a, the stamp 25 travels downwards, thus forcing the chamber/bead bed reactor 21 to bend downwards. In Fig. 2b, the stamp 25 travels upwards, thus forcing the chamber/bead bed reactor 21 to bend upwards. The beads comprised in the chamber 21 are set into vertical vibrations

The chamber/bead bed reactor comprises 21 an elastic bottom part 24a and an elastic top part 24b. The side walls of the chamber are less elastic as thy consist of a thicker material (not shown). This embodiment, together with the high filling ratio of the chamber 21, promotes the volume deformation behavior of the chamber/bead bed reactor 21 which has already been discussed, and thus increases the impact forces exerted on the cells In Fig. 3, the different possible responses of a chamber comprising beads are shown, if a force from above is exerted to the chamber. On the left side, the so called "volume displacement behavior" is shown, which is undesirable as the beads are set into horizontal movements (especially, horizontal rotations) rather than vertical vibrations, and thus result in the exertion of shear forces rather than impact forces exerted to the cells. On the right side, the so called "volume deformation behaviour" is shown, which is supported by use of stiff side walls and elastic upper and lower walls, as well as by a high filling ratio (see text). In this volume deformation principle, which is the basis of the cell lysis and/or mixing device of the present invention, vertical oscillations of the beads are promoted, which result in high impact forces exerted to the cells, and thus promote effective lysis even of rigid cells. Fig. 4 shows a microfluidic device 40 for analysis of a fluid cell sample, especially for molecular diagnostics applications, comprising a substrate 41 having a surface with a micro channel structure 42 thereon Furthermore; the device comprises a reception chamber 43 for receiving the fluid sample, wherein the reception chamber 43 is fluently connected with the micro channel 42, and a cell lysis and/or mixing device 44 according to the invention, comprising a chamber, which is filled with microparticles, and a stamp 45 which sets the chamber under vibrations.

Furthermore, the device comprises a membrane 46, which covers the upper surface of the micro channel structure arranged on said substrate in a leakage proof fashion. This results in that movement of said membrane causes a pump action on fluid located in said reception chamber in said micro channel or causes a valve action on fluid directed through said micro channel. Furthermore, the device comprises a device for actuating the movement of the membrane, namely a plunger 47.

The size of the membrane is selected so that the membrane completely covers the upper surface of the substrate 41. An up and down movement of said membrane 46 causes a pump action or valve action so that fluid located in said micro channel system, including the reception chamber, is transported in the micro channel system. An up movement of the membrane 46 causes a suction function and a down movement of the membrane 46 forces a fluid sample flow and/or causes a valve function. The plunger 47 is not in contact with the fluid sample since the membrane

46 has a fluid sealing function. The plunger 47 actuates the upper surface of the membrane 46 at specific areas so that defined areas of the membrane can be lifted up and down only.

The membrane 46 area having a valve and/or pump function is arranged adjacent and/or above the micro channel so that fluid sample in said micro channel can be forced through.

The lower surface size of the plungers corresponds with the shape of the micro channel so that a down movement of the plunger 47 contacting the membrane 46 causes a fluid pressure and/or valve action of the membrane 46. Fig. 5 shows another microfluidic device 50 for analysis of a fluid cell sample, especially for molecular diagnostics applications, comprising a reception chamber 53 for receiving the fluid sample, wherein the reception chamber 53 is fluently connected with at least one micro channel 52; and a cell lysis and/or mixing device 54 according the invention.

Fig. 6 shows another example of a microfluidic device with combined nucleic acid amplification and detection elements. The substrate 61 and the membrane (not shown), which is preferably a rubber membrane, are leak tight pressed together by a fixture of a support plate (not shown) with through going holes for receiving plungers in order to force the fluid probe by valve und pump action of said membrane through the micro channel structure to the desired area of treatment. The microfluidic device comprises a sample reception chamber 63, a cell lysis and/or mixing device 64 according to the invention, a central distribution chamber, several PCR chambers, and a lateral flow-through hybridization array with electrical contacts .

In all shown embodiments a fluid sample has to be placed below the membrane, e.g. between the upper surface of the substrate and the lower surface of the membrane. According to a preferred embodiment of the present invention, the fluid sample is placed under the membrane by means of an injection. According to an alternative embodiment of the present invention, the microfluidic device has at least one sample port for receiving a fluid sample. The receiving port can be integrated in the membrane. Preferably, the receiving port can be opened and sealed. For processing the fluid sample, it can be suitable to treat or preferably react, the fluid sample with at least one reagent. To provide a ready to use microfluidic device it may be preferred that the microfluidic device according to the present invention comprises at least one container that can release a component, for example a reagent, when opened. The container can be constructed and arranged such that it opens due to heat action and/or pressure action of the membrane. It is preferred, that the container is arranged adjacent to an area of treatment and/or adjacent to the micro channel structure so that the reagent can contact the fluid. The reagent is preferably a solid or liquid component. The liquid component can comprise a gel and the solid component can be a powder or wet powder to facilitate and speed up a reaction with the fluid sample. Fig. 7 shows an example for a chamber/bead bed reactor according to the invention, consisting of a of a bottom part (72, "lysis bed") and a top part (71, "lid"). Both parts consist of a thermoplastic material and are being produced by injection molding techniques. In the shown embodiment, micro channels 73 are implemented in the bottom part, as well as filters 74 being disposed close to the inlets or outlets. The filters are accomplished by laser drilling, with which pores can be created which have diameter of down to 90 μm.

Fig. 8 shows a model of the behavior of the particles in a vibrating bead- bed (F = 50 Hz, A = 40 % of the chamber height). The pseudo color image shows bead velocity contours and bead distribution scaled to a three dimensional 500 μm mesh.

In the case of a vibrating box, the chamber is flexible and the motion of the beads is provoked by a deformation of the box with appropriate amplitude and frequency.

The input data for the optimisation of this lysis concept are as follows: shape of the chamber (cylinder with 10 mm diameter and 3 mm height

The kind of vibrating motion is approximated by: z = z_o + A_m sin{2πft)cosfc/^r _2R) (eq. 3)

The bead bed filling ratio is about 40%, based on the theoretical consideration set forth above,

The diameter of the beads ranges between 250 and 500 μm

The main parameters to be optimised in the presented model are therefore the frequency/, the amplitude A_m.

As mentioned earlier, this configuration is very challenging for the present model. A Eulerian instead of Lagrangian approach for the modeling of the dispersed phase would be more appropriate in the frame of finite volume methods. Some first steps towards the extension of the present model to an Eulerian formulation were done during the optimization of the lysis concept described in this section: however, more development is required in this direction in order to obtain a functional Eulerian model.

In the meantime, however, the present Lagrangian approach still proved useful to identify trends and guide the optimization process. In order to model the vibrating chamber, the chamber motion needed to be built as a time dependent mesh movement. This mesh motion is fully and implicitly coupled to the solution of the unsteady Navier-Stokes equations describing the fluid through an additional "space conservation law" for the moving coordinates velocity components (Arbitrary Lagrangian-Eulerian approach).

Actually, recent experimental results show that a relatively low frequency value f coupled with a high vibration amplitude A is much more effective (in terms of bead collisions), than a low amplitude motion at high frequency.

Fig. 9 shows lysis results obtained with a device according to the invention. In the sown example, yeast cells (Saccharomyces cerevisiae) in demineralised water were lysed. Fig. 9A shows a photomicrograph of the cells before lysis, whereas Fig. 9B shows a photomicrograph of the cells after 5 min of lysis (100 Hz vibration, 35 % chamber filling ratio), and Fig. 9C shows the lysed cells at the end of the process, i.e. after 10 min (100 Hz vibration, 35 % chamber filling ratio). It is clearly visible that the yeast cells, which are known to be quite rigid, are completely disrupted at the end of the process, thus releasing their contents (i.e. nucleic acids, proteins, organelles and the like) and providing them for further analysis. To provide a comprehensive disclosure without unduly lengthening the specification, the applicant hereby incorporates by reference each of the patents and patent applications referenced above.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments.

Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.

Claims

CLAIMS:

1. A cell lysis and/or mixing device (10), comprising a) a chamber (11) having a volume, said chamber being filled with microparticles (12), and said chamber being adapted to accommodate cells; b) means (15) to put said chamber under vibrations; wherein further c) the microparticles (12) within the chamber assume between 20 and 75 % of the chamber's volume.

2. The device according to claim 1, characterized in that the chamber comprises at least one inlet (13a) and one outlet (13b).

3. The device according to any of the aforementioned claims, characterized in that the chamber comprises filters or sieves which consist of pores in the chamber walls close to the inlet (13a) and/or outlet (13b).

4. The device according to any of the aforementioned claims, characterized in that the chamber comprises an elastic bottom part (14a, 72) and an elastic top part (14b, 71).

5. The device according to any of the aforementioned claims, characterized in that the device comprises a stamp (15) which sets the chamber under vibrations.

6. A microfluidic device (40, 50, 60) for analysis of a fluid cell sample, especially for molecular diagnostics applications, comprising: a) a substrate (41, 61) having a surface with at least one micro channel structure (42, 52, 62) thereon; b) at least one detecting, controlling and/or processing element; c) at least one reception chamber (43, 53, 63) for receiving the fluid sample, wherein the reception chamber is fluently connected with at least one micro channel; and d) a cell lysis and/or mixing device (44, 54, 64) according to any of the aforementioned claims.

7. The microfluidic device according to claim 6, characterized in that it further comprises a) at least one membrane (46), wherein the membrane covers the upper surface of at least one micro channel structure (42) arranged on said substrate (41) in a leakage proof fashion, b) whereby movement of said membrane (46) causes a pump action on fluid located in said reception chamber (43) and/or in said micro channel (42), and/or causes a valve action on fluid directed through said reception chamber (43) and/or said micro channel (42), c) at least one device (47) for actuating the movement of the membrane (45), comprising pressure and/or vacuum generating means; d) wherein the reception chamber (43) is formable between the membrane

(45) and the substrate (41) .

8. Use of a device according to any of the aforementioned claims for cell lysis and/or mixing.

9. A process for lysing and/or mixing cells, wherein a device according to any of the aforementioned claims is used.

10. A process for lysing and/or mixing cells, wherein a) cells are put in a chamber which is filled with microparticles, b) the chamber is set under vibrations, wherein further c) the microp articles assume between 20 and 75 % of the chamber's volume.

11. Use of the cell lysis and/or mixing device, and/or the microfluidic device according to any of the aforementioned claims for: a) chemical, diagnostic, medical and/or biological analysis, comprising assays of biological samples such as urine, blood, serum, hair and skin samples, smear probes, biopsies, plasma, forensic samples, sperm smaples, punctations, Crusta Phlogistica, sputum; b) environmental analysis, comprising analysis of water, dissolved soil extracts, animal faeces, dissolved plant extracts; c) forensic studies; d) reaction solutions, dispersions and/or formulation analysis, comprising analysis in chemical prouction, in particular dye solutions or reaction solutions; and/or e) quality safeguarding analysis.