Patent Application
20090053689
The present invention relates to a device for processing a biological and/or chemical sample, more specifically, to a device for carrying out chemical and biological processes on a sample and a method of using said device.
The processes of separation, isolation and further purification of sample components from various starting materials is of great importance for a many laboratories working in the field of biotechnology, laboratory medicine, biochemistry, veterinary medicine, food analysis, petrochemicals, chemical synthesis and other related fields.
In the organic synthesis industry, attempts to minimize systems of chemical reactors have so far been hampered by the lack of available separation means, which can be combined with a reactor. Correspondingly in the life science industry, there is also often need for fast isolation means for a sample material due to the risk of decomposition or degradation and reliable means to prevent sample contamination. In addition, both the above-mentioned industries of science utilize common analysis methods that often require the removal of certain components, such as salts or detergents.
In order to support the above-mentioned rapidly growing industries, new techniques and methodologies are typically developed in tandem with new technical advances. These developments (both technical and methodological) generally aid in the analysis and investigation of the constituents of various cellular forms. Analysis at this level is often carried out through the investigation of a captured nucleic acid, such as for instance deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), or a protein for example originating from an organism or a cell type. In addition, to better understand the organism (or cell-type), or to isolate a compound of interest, a respective compound or organism is typically extracted using physical, chemical and/or biological interactions with different probes, chemicals, probes, and/or enzymes.
Extracting nucleic acids from various materials is known to be a tedious process. A recent evaluation report (Mattocks C, “Automated extraction methodologies”, www.ngrl.org uk/Wessex/downloads/Evaluation_of_Automated_Extraction_Methodologies—093444.pdf) gives an overview on current methods used to extract nucleic acids. The following patent applications describe devices that serve to aid in the extraction of DNA and RNA.
U.S. Pat. No. 6,649,419 describes a method and an apparatus for extracting, identifying and manipulating proteins or peptides using magnetic beads. The magnetic beads include a coating having an affinity to absorb/adsorb proteins and peptides and hence, the magnetic beads are used to separate and purify, immobilize and assay antibodies. Once the magnetic beads are bound to their respective protein or peptide, a magnetic rod is inserted to attract said beads to the rod. The binding reactions between the beads and the desired protein or peptide, and any subsequent processing reactions take place in several individual chambers and require the magnetic beads to be removed from one chamber and be physically transported, with environmental exposure, to the next chamber for further processing. Accordingly, there lies a risk of the sample or the target matter being contaminated.
US patent application 2004/0126783 describes an apparatus having a porous support, wherein said porous support includes an agent that reacts with a nucleic acid inhibitor component upon contact. The apparatus also includes a housing with a chamber defined therein. The nucleic acid component is directed through a portion of the porous support and later on, is separated from said support. The separation means of the nucleic acid from the support is carried out by a magnetic substrate means. The magnetic substrate binds to the nucleic acid and is later extracted from the sample by means of a magnetic field provide for by a magnet.
In addition to the above devices, European Patent EP 0389063 describes a method of isolating and purifying a nucleic acid from lysates of various samples. The method disclosed in EP 0389063 involves the mixing of the said lysates with a chaotropic substance and a particulate nucleic acid binding solid phase comprising silica or a derivative thereof. It is known that, in the presence of a chaotropic substance, nucleic acids are released from cells and cell lysates, and bind to silica-based nucleic acid binding solid phases. This method commonly utilizes layers of matrices of silica fibers as a binding media, and subsequent ultra-centrifugation steps are required to separate the supernatants after each washing stage. In some embodiments of the method, the centrifugation step can be eliminated, by use of commercially available silica-coated magnetic beads for binding with nucleic acid. Subsequently after binding with the nucleic acids, the sample and magnetic beads mixture are subjected to a magnetic field in which the supernatants are separated. Further examples of apparatuses for processing a biological sample have been disclosed in U.S. Pat. No. 6,562,239, U.S. Pat. No. 6,413,420, US patent application 2004/0173537 and US patent application 2005/0009071.
In each of the above-mentioned methods and apparatus used in the processing of test samples, the difficulty of preventing contamination arises. Recent advances promote the use of automated systems to perform the processing of the test sample. An example of such a system utilizes functionally coated magnetic beads for specific separation of nucleic acids, for example. However, the present systems that utilize magnetic beads in biological and/or chemical sample processing require either multiple transferences of the sample or reagent, or are unable to carry out several processing steps simultaneously or sequentially.
Accordingly, despite the above-mentioned devices and method, there still exists a need for a sample-processing device that is simple to use, capable of providing contamination-free processing of single and multiple samples, is scalable in size and yet cost-effective to produce. Such a device, and apparatus, as defined in the independent claims appended herewith, overcomes the above-mentioned difficulties.
The present invention provides a device for processing a biological and/or chemical sample, more specifically, to a device for carrying out one or more chemical and/or biological processes on a sample and a method of using said device. Thus in one aspect the present invention provides a device for processing a biological and/or chemical sample. The device for processing a biological and/or chemical sample includes at least one sample processing chamber having an inlet at a first end and a penetrable sealing layer at a second end. The sealing layer forms at least a part of an inner wall of the sample processing chamber. The sealing layer is adapted to seal off the sample processing chamber from the environment until said sealing layer is penetrated to form an outlet. The device further includes an absorption layer, wherein upon penetration of said sealing layer, said absorption layer is in fluid contact with said sealing layer. The absorption layer is capable of absorbing fluid released from said sample processing chamber, via the outlet.
In another aspect the invention provides an apparatus for processing a biological and/or chemical sample. The apparatus includes at least one device as described above and a probe. The probe is adapted to selectively attract target matter from the sample processing chamber.
In a further aspect the invention provides a method of processing a biological and/or chemical sample using the device described above. The method includes:
In yet another aspect the invention provides a further method of processing a biological and/or chemical sample. The method includes:
In yet another aspect the invention provides a fluid separation device. The fluid separation device includes a penetrable sealing layer and an absorption layer. The penetrable sealing layer is adapted to form a wall of a sample processing chamber of the device as described above. The absorption layer is in fluid contact with said sealing layer upon penetration of the sealing layer. Upon penetration of the sealing layer the absorption layer is capable of absorbing fluid released from the sample processing chamber, via the outlet.
The device for processing a biological and/or chemical sample includes at least one sample processing chamber having an inlet at a first end and a penetrable sealing layer at a second end. The sealing layer is adapted to seal off the sample processing chamber from the environment until the sealing layer is penetrated to form an outlet.
The term “environment” refers to the exterior (vs. the interior) of the sample processing chamber and therefore means any matter that is not located inside or contacting the interior of the sample processing chamber. It thus refers to any space or matter that is not in direct contact with fluid filled into the sample processing chamber while the sealing layer is intact. They are consequently not facing the environment. Hence, the environment includes further elements of the device. One such element are one or more absorption layers. At least one respective absorption layer is therefore upon penetration of the sealing layer in fluid contact with the sealing layer. In some embodiments at least one such absorption layer is directly contacting the sealing layer, for instance by means of direct physical contact. In some embodiments the sample processing chamber, the sealing layer and the absorption layer form one physical unit. In some of these embodiments both the sealing layer and the absorption layer form a wall or a part of a wall of the sample processing chamber. The absorption layer may for example be integrated into the sealing layer or arranged between two sealing layers or a sealing layer and a supportive layer. An absorption layer is capable of accommodating, soaking up, incorporating, sucking in, transmitting or otherwise absorbing or accepting fluid released from the sample reaction chamber, via the outlet that is created upon penetration of the sealing layer.
As a chamber is defined to be such that it is cut-off from the external environment as defined above, it follows that the sealing layer is affixed to the chamber in a manner to maintain a desired fluid, for instance a liquid, in the sample processing chamber, i.e. to remain impermeable for the fluid contained therein. In some embodiments the sealing layer hermetically seals the sample processing chamber from the environment. In other embodiments the sealing layer is permeable for fluid that is not of interest for a desired process to be carried out in the sample processing chamber, such as for instance gas.
In one embodiment, the sealing may occur by way of adding adhesives or sealing gels, for example to the periphery of the sealing layer to obtain an airtight fit between the sealing layer and the chamber. In other embodiments, the sealing layer may be attached to the chamber either mechanically (by pins or stitching) or by a snap-fit or tight-fit mechanism.
Accordingly, when the sealing layer is affixed to the chamber, the sealing layer forms an inner wall of or a portion of an inner wall of the chamber. As indicated above, the interior of the sample processing chamber means any space or matter that is in direct contact with fluid filled into the sample processing chamber while the sealing layer is intact. It also refers to any space or matter that may be comprised in space or matter contacting such fluid. Accordingly the term “inner wall”, when used in connection with the sample processing chamber, refers to surface areas that face the interior of the sample processing chamber in that they are able to contact fluid filled therein while the sealing layer is intact. The chamber is then capable of receiving a fluid and/or a sample, e.g. a chemical and/or biological sample, as well as optionally further matter. Hence, in some embodiments the sample processing chamber is filled with a medium, for instance a medium for carrying out a chemical and/or biological process. In other embodiments it may be desired to fill the sample processing chamber with such medium in order to carry out a chemical or biological process. The sealing layer thus acts to prevent any fluidic communication between the interior of the chamber and the surrounding environment.
As indicated above, in some embodiments the sealing layer and the absorption layer may form one common unit. They may for instance form a sealing portion, which may as a whole be penetrable. A respective sealing portion may form at least a part of a wall of the sample processing chamber. A part of or the entire inner wall of the sample processing chamber may thus for example be formed by the sealing layer comprised in a respective sealing portion. The external surface of the sealing layer may thus be adapted to seal off the sample processing chamber from the environment as defined above, until it is penetrated to form an outlet. The external surface of the sealing layer is defined in that it does not contact any fluid filled into the sample processing chamber while the sealing layer is intact, i.e. not forming an outlet. Hence, the external surface of the sealing layer is in fluid contact with the absorption layer. In some embodiments it is in direct physical contact with the absorption layer. In one of these embodiments the sealing layer forms a housing for the absorption layer. Any such embodiment of a combined sealing layer and an absorption layer may form a fluid separation device that is adapted to be used with a sample processing chamber of a device of the present invention. Such a fluid separation device is thus adapted to absorb fluid released from a sample processing chamber. It is furthermore adapted to complete a device as described above, when combined with the sample processing chamber.
Where fluid, e.g. liquid, is desired to exit the sample reaction chamber, the sealing layer will be penetrated. The penetrability of the sealing layer depends upon the properties of the material used to fabricate said sealing layer. For instance, a material with a high tensile strength or elasticity will typically have a low degree of penetrability. As the sealing layer should be easily penetrable by a user, the tensile strength of the material used should generally not be of not too high strength. The sealing layer may have, in addition to generally being of lower tensile strength, several perforations or break lines to aid in the penetration of the sealing layer. The perforations may also serve as pre-existing cracks within the structure of the sealing layer to further aid in the penetration of said sealing layer by the user.
If present, the perforations on the sealing layer are typically such that they do not run through from the internal surface to the external surface of the sealing layer in order to preserve the integrity of the sealed chamber and to prevent fluid communication between the internal surface and external surface. Instead, the perforations may, for example, only extend from the internal surface to half (or alternatively to three-quarters) of the thickness of the sealing layer. Perforating a given portion (or region) of the sealing layer causes said portion to be structurally weaker as, at a macroscopic level, some of the bonding mechanisms may be weakened. This allows the integrity of the sealed chamber to be maintained whilst easing the possible penetration of the sealing layer. Such a configuration may be particularly useful in embodiments where the sealing layer is penetrated by a probe.
Alternatively, the penetrable sealing layer may include a one-way valve. A respective valve may for instance be operable by mechanical or magnetic means. Other means to operate a respective valve include, but are not limited to, pressure, a change in temperature, and/or a change in chemical property such as pH in cases where a smart material is used (see below). As an illustrative example, a valve may include one or more elements of piezoelectric material (see below). A valve may furthermore be adapted to permit only a probe to pass through the sealing layer. In an embodiment including said one-way valve, dimensions of the valve may be such that it allows for a semi-tight fitting between the interface of the probe and that of the valve. The semi-tight fitting is necessary to allow the probe to continue to slide through said valve. Accordingly, it is to be understood that in this embodiment, the one-way valve functions, essentially, as an outlet for the chamber.
In further embodiments the sealing layer includes one or more elements that are able to disintegrate or that cause other parts or elements of the sealing layer to disintegrate. Such elements form an outlet or generate an outlet. This may be achieved by means including, but not limited to, pressure, mechanical means, moisture, an electrical field, a magnetic field, a change in temperature, and a change in pH of the fluid comprised in the sample processing chamber, or any combination thereof. In some embodiments respective properties of a sealing layer are achieved by including a smart material. The term “smart”, or responsive, material is used in the art for materials that react to their external environment by bringing on a desired response. These materials thus possess a property that can be significantly altered in a controlled manner. External factors that may be suitable to alter respective properties include stress, temperature, moisture, pH, electric or magnetic fields. Any respective material can be included into the sealing layer of the present invention. Examples of suitable smart materials include, but are not limited to, piezoelectric materials (see below), thermoresponsive materials, shape memory alloys, shape memory polymers, and pH sensitive materials.
Shape memory alloys and shape memory polymers can hold different shapes at various temperatures. An illustrative example of a respective polymer is Pluronic F127. Solutions of 14-30% Pluronic F127 are fluids at low temperatures and gels at room temperature. As a further example, an aqueous solution of a poly(ethylene glycol-b-[DL-lactic acid-co-glycolic acid]-b-ethylene glycol) (PEG-PLGA-PEG) triblock copolymer forms a free-flowing sol at room temperature and becomes a gel at body temperature. A further example are phenylboronic acid-poly(vinyl alcohol) hydrogels, which swell when glucose binds to them.
An illustrative example of a pH sensitive material is poly(2-hydroxyethyl-methacrylate) (PHEMA), which swells in basic solution and shrinks in acidic solution. An example of a material that is sensitive to an electric field is a semi-interpenetrating polymer network (semi-IPN) hydrogel, composed of chitosan and poly(hydroxyethyl methacrylate) (PHEMA, see above). When in contact with aqueous NaCl solutions, this material swells. If the material is exposed to an electrical field in this state, it shows a bending behaviour. Two illustrative examples of a temperature sensitive material are poly(N-isopropylacrylamide) (pNiPAAm) and poly(N,N-diethylacrylamide) (PDEAAm). These materials undergo a reversible swelling within a defined temperature range. The swelling is accompanied with a change in volume and hydrophobicity. The change of pNiPAAm is further sensitive to the pH of a solution, which is in contact with it. Where desired, some smart materials such as pH-sensitive materials or chromogenic materials may also be used for indicator purposes in the device of the invention. Chromogenic materials change colour in response to electrical, optical or thermal changes.
As an illustrative example for a respective sealing layer, the sealing layer may contain a wax. Such a wax may melt when subject to its respective melting temperatures. As a further illustrative example, is for instance common in the art to characterize polymers by the dependence of their viscoelastic properties on the temperature. Typically mechanical properties of a polymer change at the crystalline melting temperature. As an illustrative example it may be desired to perform a process in the device at a certain temperature. Given this temperature, polymers can be identified, which posses a crystalline melting temperature close to the chosen temperature. A part of the sealing layer may then consist of this polymer. The polymer will stand the reaction conditions, however a further change in conditions may easily cause the polymer to change its aggregation state. After the process has reached a desired degree of completion, a change in pressure may for instance be applied to cause an opening of the sealing layer at the respective site. An example of a polymer of a very low crystalline melting temperature is polydimethylsiloxane with a Tm of −40° C. An example of a polymer with a crystalline melting temperature close to room temperature is natural rubber (polyisoprene) with a Tm of 28° C. As yet another example, a thin layer of an intrinsically brittle polymer, such as, but not limited to, polystyrene and poly(vinyl chloride), is easily susceptible to pressure or mechanical force. Where desired, a so called “rigid-rod” polymer may be employed. Rigid-rod polymers are polymers, which do not undergo a change of polymer conformation, although the primary backbone of the polymer is deformed by applied stress. Examples of a device possessing a sealing layer that is able to disintegrate in its entirety are depicted in
If present, the shape of perforations may vary according to the shape of the probe being utilized to create the outlet. Should it be desired to use a probe which is generally cylindrical in nature, an asterisk shape perforation may be used (cf. e.g.,
Alternatively, a hinged design may be employed in that instead of having a complete perforation of a square, for example (as depicted e.g. in
Using the above-mentioned embodiment of a square with three perforated sides, a further alternative may be to have a notch with the acute portion of said notch extending upwards from the external surface of the sealing layer towards the internal surface (cf.
In some embodiments the sealing layer contains a recess. The recess may be of a larger diameter than the spatial extent of the penetrating portion of the probe in the same dimension. In some of these embodiments, the recess is present on the side of the sealing layer that forms part of the internal wall of the chamber. The recess may be such that it contains only a very small volume of fluid. The recess may, for example, be selected to be able to accommodate a volume of less than 10 μl. The small recess may also be adapted to fit to the probe such that when the probe is inserted into the chamber and over the recess, the probe may form, in conjunction with the recess, a mini-reaction chamber within said chamber. (cf.
The sample reaction chamber may include any desired material. Typically, walls of the sample reaction chamber are solid and able to remain intact during the entire process to be performed therein. The sample reaction chamber may be of any desired volume. It may for example be able to accommodate a volume of 0.1 μl to 100 ml, or a volume of 1 μl to 10 ml, or 10 μl to 2 ml. Where fluid is filled into the chamber, only part of the volume of the sample reaction chamber may be filled with the respective fluid. The remainder of the chamber volume may for instance be occupied by other fluid, such as air or an inert gas.
The inlet of the sample processing chamber may consist of or may include any means. As an illustrative example, the inlet may be an opening. Such an opening may be of any size and shape. Examples include, but are not limited to, the shape of a circle (cf. e.g.
In some embodiments the inlet of the sample processing chamber is adapted to receive a probe. As an illustrative example, the inlet may be a hole in a wall of the sample processing chamber, which is adapted to allow for a probe to enter. In such embodiments an interface, for instance a void space, may remain between the probe and a brim or edge of a respective hole. Such an interface between the probe and the wall of the chamber, covering the remaining size of the inlet, may be sealed by means of a sealant such as a rubber ring, a viscous gel or an epoxy material. Essentially, any sealing means that permits the probe to slide into the chamber and penetrate the sealing layer may be used.
When used for the processing of matter (e.g. a sample), the device may be subjected to an energy source for particular processes to be initiated or catalyzed. Examples of energy that may be applied, include, but are not limited to, microwave or photolytic energy. Accordingly in a further embodiment, the chamber of the device may be adapted to have a surface with waveguides that transmit the microwave or photolytic energy from the source towards the sample in the sample processing chamber. In addition to the above, in some embodiments piezoelectric materials are attached to the side walls of the chambers. Piezoelectric materials reversibly acquire a charge when compressed, twisted or distorted. Conversely they therefore change dimensions in a reversible manner, when exposed to an electric field, an effect termed electrostriction. These properties can be utilised for the generation of motion, respective devices are termed electro-mechanical transducers or piezo actuators. Examples of suitable piezoelectric materials include, but are not limited to, berlinite (AlPO4), gallium orthophosphate (GaPO4), barium titanate, lead titanate, lead zirconate, lead zirconate titanate (Pb[Zr,Ti]O2), lead magnesium niobate, and polyvinylidene fluoride (—CH2—CF2—)n.
The device may be configured for the processing of only one or a plurality of matter, such as for instance test samples, at the same time. In this respect, the sample processing chamber may be configured to accommodate or contain sub-chambers separated from each other and to be loaded with any target matter, e.g. one or more test samples. In some embodiments the device is adapted to provide a particular interior geometry for a desired reaction, for instance for the exposure of a plurality of test samples or only one sample to microwave or photolytic energy. The configuration aims to achieve a uniform distribution of the microwave or the photolytic energy for uniform photo activation or uniform heating of the contents of the chamber, for example.
In some embodiments the sample processing chamber may be adapted to include one or more sub-chambers (such as for instance a basket), which may for example be loaded with one or more test samples. In embodiments where the sample processing chamber includes such sub chamber(s), these sub-chamber may have a sealing layer as described above. In such an embodiment, the sub-chamber may be placed within the sample processing chamber along with a sample and/or requisite reagents for the reaction to be carried out. Accordingly, a first reaction may be carried out in said sub-chamber and the resulting contents of said first reaction may then be transported (via the penetration of the sealing layer) to the sample processing chamber of the device for further processing. A sub-chamber may located anywhere within a sample processing chamber. As two illustrative examples, they may be stacked within the sample processing chamber as shown in
In some embodiments the sample processing chamber has a thermostatic device or a Peltier heater incorporated therein to influence (by way of acting as a catalyst, for example) the processing of the chemical or biological sample by varying the temperature. In this regard, the thermostatic device or Peltier heater may act a heating means, a cooling means or both. Accordingly, the heating or cooling means may be, but is not limited to, a peltier heater attached to the surface or surfaces of the chamber, or a heating or cooling coil wound around the circumference of the chamber, for example to provide for uniform temperature distribution. A respective thermostatic device may be located anywhere in the sample processing chamber. Alternatively, when the device of the invention is used in connection with a probe, such a probe may include the thermostatic device.
In further embodiments, the device is adapted to be securable to at least one other device, such that the outlet of one device is in fluid communication with the inlet of another device. The securing means between any two devices may for instance be achieved by having corresponding slots and rails by which one device may be secured to another. Alternatively, a socket and plug configuration may be utilized as well. In the socket and plug embodiment, the base (or any other) portion of the device may include a socket. The corresponding or mating surface is thus capable of functioning as a plug, for example. A further alternative in securing at least two devices together may be achieved by using snap-fit or tight-fit means. In yet other embodiments one device is screwed onto another. Further methods of securing at least two such devices together may be used provided the guiding principle of maintaining the outlet of one device in fluid communication with the inlet of a subsequent device is adhered to. It should be understood that a single device of the present invention may furthermore contain more than one sample processing chamber (cf. e.g.
For each of the above-mentioned securing means, an arrangement of a plurality (which refers to two or more) of devices may be carried out in series in any (one or more) dimensions. As an illustrative example, devices may be arranged vertically or horizontally. In a vertical arrangement, essentially, stacking takes place. The stacking may be collinear or in a step-like manner. Should a horizontal arrangement be used, again, the arrangement may be collinear or step-like but in a horizontal orientation. Illustrative embodiments of horizontal and vertical arrangements are depicted in
In stacking the devices, as mentioned above, alternatively an external modular mechanism may be used. Apart from supporting the stack, the external modular mechanism may also be adapted to function in conjunction with additional means used in a desired process. An illustrative example is medical diagnostic equipment. The external modular mechanism may for instance have an opening or slot adapted to accommodate the device of the present invention. Where for instance some medical diagnostic equipment is comprised in the external modular mechanism, such equipment may for example allow the user to select a suitable probe, which includes certain characteristics (such as for instance antibody coatings). It may furthermore allow a user to insert such suitable probe to perform a specific test. The external modular mechanism may also include a security mechanism that assists in securing the position of stacked devices to other medical devices.
In any of the embodiments described herein, the sealing layer may be adapted to be penetrable by mechanical means. In this regard, the above mentioned perforations may be used to adapt said sealing layer to be penetrable by mechanical means.
In other embodiments, the penetrable sealing layer and/or the absorption layer may be detachable. The sealing layer may be detachable from the outlet. It may furthermore be replaceable with at least one other penetrable sealing layer. The absorption layer may be detachable from the sealing layer. It may furthermore be replaceable with at least one other absorption layer. In such embodiments, the sealing layer may be adhered to the chamber or the absorption layer to the sealing layer by means of, but not limited to, a removable adhesive such as double-sided tape, a temperature dependent epoxy adhesive or an adhesive putty such as that from 3M (3M Scotch Removable Adhesive Putty), for example. Alternatively, the sealing layer may also be adhered to the chamber or the absorption layer to the sealing layer by mechanical means such as screw on adaptation, latches, tight-fitting or snap-fitting, for example. A respective replacement of a sealing layer or an absorption layer may be desired where a device that has already been used for performing a process therein is desired to be reused.
A detachable sealing layer or absorption layer may be advantageous in a number of uses of the device. This may for instance be due to the fact that the user may simply replace a sealing layer, which may for instance be penetrated or disintegrated with a new sealing layer. Likewise an absorbing layer, which may for instance be soaked with a fluid, may be exchanged for a new respective sealing layer. Where desired, this enables the user to reuse the chamber thereby reducing waste and costs.
As indicated above, in other embodiments the device may further include a second penetrable sealing layer. In some of these embodiments the first and the second sealing layer are arranged to sandwich the absorption layer between them. The second sealing layer may provide additional mechanical support to the absorption layer sandwiched there between. In some applications, the second sealing layer can also serve as a barrier to prevent contact of the absorbed fluid in the absorbing layer from in contact with the atmosphere particularly when the fluid maybe health hazard. The second sealing layer, as in the first sealing layer, may also be adapted to be penetrable by means of the above-mentioned perforations.
As indicated above, in some embodiments of the device, the penetrable sealing layer includes a recess. A head of a probe may enter into such a recess up to the given depth of said recess without penetrating the sealing layer (cf.
The sealing layer(s) of the device of the invention may comprise any material that is able to perform a sealing function under the conditions intended for its use. Where desired, the material may be selected to withstand certain reaction conditions that are intended to be used. Where for instance corrosive matter is used in a process performed in the sample processing chamber, the surface of the sealing layer that faces the interior of the sample processing chamber may be equipped with or consist of material that is inert to such matter. In other embodiments the sealing layer may be used to terminate a reaction. As an illustrative example a sealing layer of a certain material and with a certain strength may be known to disintegrate under selected reaction conditions after a defined period of time. In this case the strength of the sealing layer may be adapted to stand a desired time interval, where after it disintegrates thus releasing fluid from the sample processing chamber. This may cause the termination of a reaction occurring in the sample processing chamber.
Examples of materials that are typically suitable for the use of aqueous fluids include, but are not limited to, inorganic materials and natural or synthetic polymers. Examples of suitable polymers include, but are not limited to, polypropylene, polyisoprene, polystyrene, polyvinyl chloride, polyisobutylene, polyethylene terephthalate (PET), polyacrylates (e.g. polymethyl-methacrylate (PMMA)), ethylene-vinyl acetate (EVA) copolymers, phenol formaldehyde resins, epoxy resins, poly(N-propargylamides), poly(O-propargylesters), and polysiloxanes. Two illustrative examples of inorganic materials that may be used under most conditions are glass and a metal foil. A respective metal foil may for instance be an aluminium foil. A metal foil may optionally be coated with a polymer layer such as polyethylene or polypropylene, for example were it is desired to avoid oxidation.
The absorption layer may be designed to accommodate, soak up, incorporate, suck in, transmit or otherwise accept or absorb any volume of a selected fluid. It may thus for instance be designed to absorb any part or the complete volume of the sample processing chamber as well as of any other chamber other of the device. The absorption layer is in some embodiments furthermore designed to be penetrable.
In one embodiment, the absorption layer of the device may include at least two sub-absorption layers. In one embodiment having two absorption layers, the second absorption layer may be the same as the first in order to further increase the absorbance capacity of the absorption layer as a whole. In the embodiment having two similar absorption layers, should the volume of medium be too large for the first absorption layer to absorb completely, the subsequent absorption layer may also aid in absorbing any excess fluid not already absorbed by the first absorption layer, for example.
Alternatively, the absorption layer may include sub-absorption layers of varying absorptivity. The varying absorptivity may be of advantage when the medium contains fluids of varying volatility, viscosity or miscibility. If the medium contains two immiscible fluids, for example, the first absorption layer may be only capable of absorbing one of the two fluids leaving the second absorption layer to absorb the remaining fluid. Accordingly, two fluids of varying volatility may also makeup the medium. As above, it may be the case that a particular absorption material is used to absorb only one of the two fluids, for example.
As mentioned above, an alternative embodiment of the absorption layer utilized may have an affinity or a selectivity for absorbing particular fluids. Accordingly, in such an alternative embodiment the adsorption layer may be fabricated from a hydrophilic material or a hydrophobic material. Such material may for instance be used in form of a fluid-absorbing polymer powder or a polymer gel. In embodiments where the absorption layer is fabricated from a hydrophilic material, the hydrophilic material may for instance be based on a polysaccharide such as for instance cellulose. Such cellulose-based materials may be selected from, but are not limited to, the group consisting of a wood pulp fiber, a cotton linter, rayon fibers, flax, hemp, jute, ramie and cotton, for example. A cellulose fiber material well known in the art is commercially available as Whatman paper. Other fluid-absorbing polymers that may also be used in the fabrication of the absorption layer include, but are not limited to, cellulose, starch, polyurethane foam and poly(vinyl amine)s. Examples of hydrophobic absorption materials include, but are not limited to, natural, synthetic, and modified natural polymers. Three illustrative examples are cellulose fibers, which are coated with an hydrophobic compound, perfluoropolyether fibers and polypropylene fibers. Hydrophobic absorption materials are commercially available, for instance under the trade names K-Sorb Hydrophobic®, OCLANSORB, IRGASURF, and KLAUSORB.
For some applications it may be desired to provide an absorption layer that is at the same time both of a large capacity and designed to be easily penetrated. A large capacity may require a significant thickness, while an easy penetration may require thinner material. In some embodiments of the device the absorption layer is therefore of a non-uniform thickness. The absorption layer may for example posses one or more recessed regions at the surface opposing the sealing layer as illustrated in
In other embodiments the absorption layer is made up of just one homogenous layer, which may be of uniform absorptivity. In any of these and the aforementioned embodiments, the absorption layer (e.g. a homogenous absorption layer) may be adapted to have sufficient capacity for absorbing a fluid of volume equating to the volume of the chamber. In other words, the absorption layer may be able to absorb a chamber-full of fluid.
The absorption layer contains in some embodiments fluidic means that direct any fluid, which is released from the sample processing chamber during penetration of the sealing layer, to a physical space. As an illustrative example, the absorption layer may contain or consist of a material that shows little absorptivity, but that is able to transfer fluid to a fluid line that direct it to a discharge unit. Examples of a respective fluid line include, but are not limited to, a channel or a tube. An embodiment of such an absorption layer is illustrated in
In a further embodiment, the entire device may be placed within a housing. The housing may also be adapted to take a plurality of said devices. Additionally, during the arrangement of a plurality of said devices, the housing may allow said plurality to be further secured together. Alternatively, the housing may be a raised platform or a means of further isolating the device in order to reduce and prevent the occurrence of contamination to samples during processing.
As mentioned above, the device may contain more than one sample processing chambers. In another embodiment, the device may thus include two, three, four or more sample processing chambers as for example illustrated in
All the above-mentioned embodiments of the device may be implemented in an apparatus for processing a biological and/or chemical sample. Accordingly, the present invention also refers to an apparatus for processing said samples. The apparatus includes at least one device, as described in any of the aforementioned embodiments, and a probe, wherein said probe is adapted to assist in the processing of the sample. As an illustrative example, the probe may selectively attract target matter from the sample processing chamber. Another illustrative of assisting in the processing of the sample example of regulating the temperature of fluid or the sample in the sample processing chamber. Typically, but not necessarily, the probe can be inserted into and removed from the sample processing chamber via the inlet of the device if required.
The probe may be designed to serve the function of attracting target matter through means such as physicochemical means or physical fields and forces such as for instance magnetic and electromagnetic fields or electrical charges. Three illustrative examples of attractions by physicochemical means are van-der-Waals interactions, ionic attractions and affinity attractions such as for instance antibody-antigen binding attractions. As described below, target matter may bind to the probe directly or via additional means, i.e. indirectly. Any part of the probe may be designed to attract certain target matter. As an illustrative example the probe may posses a head (head of probe), which, or a part of which, includes magnetic matter or provides a surface with affinity for certain target matter.
Two illustrative examples of a probe depicted in
In some embodiments binding and release of target matter to and from the probe is controlled through a change of the properties of the probe. An illustrative example is the change of magnetic properties of the probe as for instance illustrated above and in
An attraction or binding of target matter to the probe may be achieved by any means. It may in some embodiments be desired to select means that do not affect, change, spoil or damage the target matter. In other embodiments the means of attracting or binding target matter may be selected to be part of a desired process to be performed in the device of the invention.
In one exemplary embodiment, the probe contains a substrate capable of binding target matter such as, but is not limited to, DNA, RNA, a synthetic analogue thereof, a protein, bacterial cells or spores, viruses, or low molecular weight organic molecules. The substrate thus has a higher affinity to the target matter than to other matter. The probe may for instance be coated with a respective substrate. Examples of a respective substrate include, but are not limited to, an antibody, a fragment thereof and a proteinaceous binding molecule with antibody-like functions. Examples of (recombinant) antibody fragments are Fab fragments, FV fragments, single-chain FV fragments (scFV), diabodies or domain antibodies (Holt L J et al., Trends Biotechnol. 21(11), 2003, 484-490). An example of a proteinaceous binding molecule with antibody-like functions is a mutein based on a polypeptide of the lipocalin family (WO 03/029462, Beste et al., Proc. Natl. Acad. Sci. USA 96, 1999, 1898-1903). Lipocalins, such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or glycodelin, posses natural ligand-binding sites that can be modified so that they bind to selected small protein regions known as haptens. Examples of other proteinaceous binding molecules are the so-called glubodies (see WO 96/23879), proteins based on the ankyrin scaffold (Hryniewicz-Jankowska A et al., Folia Histochem. Cytobiol. 40, 2002, 239-249) or crystalline scaffold (WO 01/04144,) and the proteins described in Skerra, J Mol. Recognit. 13, 2000, 167-187. In addition the probe may be coated with a modifying agent that further increases the affinity of the substrate for any or a certain form, class etc. of target matter.
In another exemplary embodiment, the probe is able to attract a substrate. The substrate in turn is capable of attracting or binding target matter. Magnetically attractable particles (functionalized or otherwise) used to capture the target matter are an illustrative example of such a substrate. In such an embodiment, the probe design may provide a hollow primary core rod (sheath) (cf. e.g. sheath 25 in
For convenience magnetically attractable particles are herein referred to as “magnetic particles” or “magnetic beads”. Magnetic particles may contain ferromagnetic or supermagnetic material. Supermagnetic material responds to a magnetic field with an induced magnetic field without a resulting permanent magnetization. Magnetic particles based on iron oxide are for example commercially available as Dynabeads® from Dynal Biotech, as magnetic MicroBeads from Miltenyi Biotec, as magnetic porous glass beads from CPG Inc., as well as from various other sources such as Roche Applied Science, BIOCLON, BioSource International Inc. or Novagen Inc., to name a few. Magnetic nanoparticles based on supermagnetic Co and FeCo, as well as ferromagnetic Co nanocrystals have been described, for example by Hütten, A et al. (J. Biotech. (2004), 112, 47-63).
As mentioned above, an alternative embodiment may allow a secondary core rod to be inserted into a sheath (hollow primary core rod) to form the probe (cf. e.g. core rod 16 in
In other embodiments the probe may simply consist of a long rod ending in a head (which may serve in binding target matter) as depicted in
The core rod and any arms, blades etc. attached thereto may also serve the function of penetrating the penetrable sealing layer (and absorption layer, if necessary) to form the outlet for the fluid to depart the chamber. Accordingly, the departing fluid is absorbed by the absorption layer in fluid contact with said sealing layer. In embodiments of the device where at least two devices are in fluid connection with each other (either via stacking or horizontal arrangement), the probe, with the attached sample substance, may be used to transfer said matter from one chamber to another for further processing without any exposure to the environment.
In a further embodiment, the arms of the probe may be adapted to serve as a protection for a part of the probe. As an illustrative example arms of the probe may protect a head of the probe to prevent target matter attached thereto from brushing off by the broken edges of the penetrated sealing layer. In such an embodiment, the ends of the arms may extend beyond the head of the probe.
In some embodiments, the arms may have single or multiple channel(s) embedded in them. In one such embodiment the channels are in fluid connection with external fluid reservoirs from the environment. Accordingly, said channels may be used for dispensing fluids into the chamber of the device, which is otherwise sealed of from the external environment. The connection to the external environment may be pressurized thereby enabling the probe to also have a suction and dispensation means to enable it to mix, stir etc. any fluid and other matter within the chamber to ensure a uniform mixture where needed. In such an embodiment, the probe may be also adapted to introduce and remove the test sample into the sample processing chamber thereby reducing the risk of contamination, as the probe would remain within the chamber from the very beginning.
The channels may also be used for dispensing sealants into the chamber of the device. The sealant may be room temperature curing polymers, heat curing polymers, or photo-curable polymers, for example. In this regard, the sealant may be dispensed at any stage of the processing sequence but it may be preferably carried out in the last stage. As an illustrative example, in the extraction of nucleic acids, the last step is the elution step. In said elution step, a silicon sealant is dispensed on top of the collecting tube or recess wherein the target matter was located. The purpose of dispensing a sealing layer is a reduction of the risk of exposing a sample to contamination while collecting it. As an illustrative example, an arrangement as depicted in
The probe may possess arms of the dimensions shown in e.g.
In a further embodiment, arms, blades or other elements surrounding the core rod, such as a hollow cylindrical structure for example, serve as a means of protection for matter that is attached to the probe, as illustrated in
The probe is in some embodiments adaptable to various embodiments of the devices, such as different dimensions of the chamber, different sizes of recesses of the sealing layer etc. As an illustrative example, a probe may possess diametrically translatable arms. Such a probe is thus in some embodiments able to alter the distance between a central head and the translatable arms. Such a probe may furthermore be able to alter its diameter relative to the sealing layer. This may for instance result in a variable penetration area in the sealing layer, which allows the invention to work for different volumes in each chamber. In this regard, the invention may be suitable for use in mixing very small volumes, such as for instance volumes in the range from 5 μl to 10 μl, effectively.
In other embodiments, the probe or a part of the probe, with a sample bound to it, may be mechanically adapted to perform vertical movements, as described above. As an illustrative example, the head of the probe may be able to move vertically independently from the outer sheath and the arms. The head may, by vertically travelling a certain distance, expose matter, for instance a sample, to a fluid or a specialised compartment present in the chamber. An exposure to a fluid may result in the formation of a suspension or a solution. As a consequence a reaction with reagents present inside the chamber (e.g. dissolved in the fluid) may occur. Where a specialised compartment of the chamber contains reagents, a reaction may likewise occur. After a respective reaction has reached a desired degree of completeness, matter such as a sample may be recaptured onto a surface of the probe, for instance again to the head of the probe. Thereafter the head may retract vertically again until it re-enters the sheath of the probe.
In some embodiments of the apparatus, the probe is furthermore able to create the outlet of the chamber by penetrating the sealing layer. The probe may achieve this by having one or more pointed tips or sharpened edges, or alternatively, the probe may include a screw-like design such as that of a thread by which it may “drill” through the penetrable sealing layer.
In other embodiments the probe itself need not provide any particular design that assists the penetration of the sealing layer. In such embodiments the probe may posses an end of any design. Examples include, but are not limited to a flat end, a rounded end or a chiseled end. In such embodiments the penetration of the sealing layer may be achieved by means other than the probe, such as pressure, mechanical means, a change in temperature, and a change in pH (see above). In other embodiments the penetration of the sealing layer may be achieved by pure force, such as, but not limited to, mechanical force. In some of these embodiments the sealing layer includes a design that assists a penetration, for example by a probe, as described above.
Any part of the probe may be designed to penetrate the sealing layer of the device. As an illustrative example, in some embodiments a central rod of the probe ends in a head that juts other parts of the probe and will thus penetrate a sealing layer, especially where the sealing layer is of flat design (cf. e.g.
In other embodiments, the probe is adapted to agitate a fluid (or medium) contained within the chamber. The agitation may be in the form of a vertical, horizontal, rotational or a combinatorial displacement thereof of the fluid within the chamber. In order to achieve such a displacement, the probe may be adapted to have an impeller, vanes, a propeller or arms mounted on the portion of the probe within the chamber.
In further embodiments, the probe may have magnetic properties. Some embodiments of a magnetic probe are used in conjunction with embodiments of the device having magnetic particles contained within the chamber. In some of these embodiments matter such as reagents is coated onto suspended magnetic particles in the fluid. The magnetic properties may be induced through an electromagnet or by using permanent magnets integrated into the probe, as previously mentioned.
In yet another embodiment, the motion (rotation and penetration) of the probe may be mechanized. In such a mechanized setup, a single device or multiple devices may be arranged in series or in parallel with each other. The arrangements allow for single or multiple processes to be conducted simultaneously and/or sequentially across the different devices. In the above embodiment in which the device is incorporated into a biomedical device, such mechanization may also be implemented therein.
In a further embodiment, the device or the apparatus may include an elution chamber (cf. e.g.
In other embodiments, multiple arms or blades etc. are arranged with respect to the elution chamber such that during the elution step, only the head of the probe, but not the arms, is inserted into the elution chamber. As an illustrative example, the probe depicted in
In another embodiment, a magnetic field can be applied by positioning one or more magnetic bars or magnetic rods externally and/or internally in the device in near proximity to the probe to speed up the release magnetic particles from the probe during the dispersing process. The magnet field can be activated and deactivated between a collecting position and a releasing position. As an illustrative example, a spacer as depicted in
The device of the invention may be used for any analytical or preparative purposes or combinations thereof. An illustrative example of an analytical application is determining the effect of chemoattractants on a microorganism. Methods that determine such effects are usually referred to as a “cell migration assay”. For carrying out such a method the probe may include a respective chemoattractant, which may optionally be covered by a membrane of a defined pore size. Such a membrane may for instance be coated with components (e.g. fibronectin) that provide assistance for a microorganism in adhering to the probe. Where the respective microorganism adheres to the probe, it may, after a defined period of time, be further processed as generically illustrated below. The microorganism may be analysed while adhering to the probe or after a release. Methods of releasing cells from a substrate are well known in the art and include for instance the use of calcium-chelating compounds or the application of force, for example centrifugation.
The device is capable of processing matter by the performance of any desired method. Examples include, but are not limited to, a chemical reaction, a component exchange, disintegration, and disengagement and any combination thereof. An illustrative example of a disintegration is a cell lysis. Two illustrative examples of a disengagement are an extraction of a molecule from an organism or a part of an organism and a release of a molecule from an organism. An illustrative example of exchanging components of a sample is a washing process. At the beginning and at the end of a washing process target matter or a precursor thereof, which may be comprised in the sample, is typically bound (whether directly or indirectly via for example magnetic beads, see above) to the probe. The transfer of target matter into a fluid, its release into the fluid, its mixing with the fluid (for instance by agitation), and its subsequent recovery, make up exemplary steps of a washing process.
In some embodiments the target matter is a molecule that is suspected or known to be present within other (undesired) matter, from which it needs to be extracted. Extraction of a molecule from an organism or a part of an organism may for instance include the usage of a compound that facilitates the transfer of a desired molecule from an organism or a part thereof into a fluid. An illustrative example of an extraction of a molecule from a part of an organism is an extraction of proteins (wholly or partly) integrated into the cell membrane. It is often desired to transfer such proteins into an aqueous solution for further processing. A compound that facilitates the transfer of such proteins into an aqueous solution is a detergent. Contacting a respective cell membrane with an aqueous solution, to which a detergent is added, will typically result in an extraction of membrane proteins.
Any chemical reaction may be performed within a device of the present invention, as long as the device remains essentially intact while the reaction is performed. For some chemical reactions it may thus be required to select (a) material(s) for the surfaces of the device that possesses a particular resistance to stress occurring during the reaction, such as for instance heat or corrosive properties of the target matter, reactants, products or intermediate products. Examples include, but are not limited to, a protein synthesis, a nucleic acid synthesis, a peptide synthesis, and enzymatic degradation, an interaction with a binding molecule or any combination thereof.
The sample may be of any origin. It may for instance, but not only, be derived from human or non-human animals, plants, bacteria, viruses, spores, fungi, or protozoa. Accordingly, any of the following samples selected from, but not limited to, the group consisting of milk soil, a blood sample, a serum sample, a plasma sample, a urine sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a milk sample, an amniotic fluid sample, a biopsy sample, a needle aspiration biopsy sample, a cancer sample, a tumour sample, a tissue sample, a cell sample, a cell lysate sample, a virus culture sample, a crude cell lysate sample, a forensic sample, an archaeological sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, or any combination thereof may be processed by the device. Where desired, a respective sample may have been preprocessed to any degree. As an illustrative example, a tissue sample may have been digested, homogenised or centrifuges prior to being used with the device of the present invention. The sample may furthermore have been prepared in form of a fluid such as a solution. Examples include, but are not limited to, a solution or a slurry of a nucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide, an amino acid, a protein, a synthetic polymer, a biochemical composition, an organic chemical composition, an inorganic chemical composition, a lipid, a carbohydrate, a combinatory chemistry product, a drug candidate molecule, a drug molecule, a drug metabolite or of any combinations thereof. Further examples include, but are not limited to a suspension of a cell, a virus, a microorganism or of any combinations thereof. It is understood that a sample may furthermore include any combination of the aforementioned examples.
Often, but not necessarily, the sample will include, or will be expected to include, the target matter or a precursor thereof. Such embodiments shall be illustrated by a number of examples: The target matter may for instance be a cell or a molecule added to or comprised in the sample, and it may be desired to obtain it in a purified or enriched form. As another example, the target matter may be a compound known or theorized to be obtainable from a precursor compound by means of a chemical process. In this case the sample may for instance include a solution of such precursor compound. As yet a further example, it may be desired to remove a contaminant from a fluid, for instance from a biological sample. In this case the device may be used for decontaminating or analysing a sample and the contaminating matter is understood as the target matter. As an illustrative example, a cell culture media may be suspected to be contaminated. In this case it may be desired to identify the type of contaminant and to use the device of the invention for this purpose. The probe possesses in such embodiments an affinity to the contaminant or it is able to bind other matter that has an affinity to the contaminant.
The target matter or precursor thereof may thus be of any nature. Examples thus include, but are not limited to, a nucleotide, an oligonucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide, an amino acid, a protein, a synthetic polymer, a biochemical composition, an organic chemical composition, an inorganic chemical composition, a lipid, a carbohydrate, a combinatory chemistry product, a drug candidate molecule, a drug molecule, a drug metabolite, a cell, a virus, a microorganism or any combinations thereof. In embodiments where the target matter is for example a protein, a polypeptide, a peptide, a nucleic acid, a polynucleotide or an oligonucleotide, it may contain an affinity tag. Examples of affinity tags include, but are not limited to biotin, dinitrophenol or digoxigenin. Where the target matter is a protein, a polypeptide, or a peptide, further examples of an affinity tag include, but are not limited to, oligohistidine, polyhistidine, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG′-peptide, the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein (MBP), the HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virus glycoprotein D, the hemagglutinin (HA) epitope of the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala and the “myc” epitope of the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu. Where the target matter is a nucleic acid, a polynucleotide or an oligonucleotide, an affinity tag may furthermore be an oligonucleotide tag. Such an oligonucleotide tag may for instance be used to hybridize to an immobilized oligonucleotide with a complementary sequence. A respective affinity tag may be located within or attached to any part of the target matter. As an illustrative example, it may be operably fused to the amino terminus or to the carboxy terminus of any of the aforementioned exemplary proteins.
The device of the invention may be used manually, automatically or in a combination thereof. An automatic use may include a control system that induces in a programmed manner the performance of any acts desired for a process to be carried out using the device of the invention. Examples include, but are not limited to, motion of a probe into or within the device, pumping, injecting, stirring, the application of energy and the start of a detection machine. The use may be an integrated step of, for instance, a method of processing (e.g. isolating, reacting etc.) one or more samples. Such a method may include a number of sequential steps. As an illustrative example, the device may be used for the isolation of a desired cell population from a blood or tissue sample. An initial step of a method of processing a respective sample may be aimed at a volume reduction and include for example centrifugation. A late step of a method of processing a respective sample may include the detection of desired cells, for instance by means of an automated cell counting device. As a further illustrative example, the device may be used to perform a chemical reaction. Where the reaction product is a liquid of suitable boiling point, a late step of its manufacture may include a distillation process. For this purpose an obtained reaction mixture may be heated. The use of the device may also form a complete process, all steps of which are carried out within the device of the invention.
Thus the device is in some embodiments for instance an external, internal or integrated part of a larger apparatus. A respective larger apparatus may include any desired component. It may for example include one or more components that serve in keeping any element of the device of the invention in a desired position or one or more components that assist or serve in moving any element of the device of the invention into such a desired position. An example of such a larger apparatus that includes the device of the present invention is shown in
In this respect the present invention also provides a method for processing a biological and/or chemical sample using the above described device, the method comprising the following steps:
In some embodiments, the method further includes in step (b) providing target matter or a precursor thereof, as well as in step (c) placing the target matter or precursor thereof into the sample processing chamber of the device. As already indicated above, in other embodiments the target matter is included in the biological and/or chemical sample. In some embodiments, the method further includes:
In an exemplary illustration of the use of the apparatus, a reaction involving magnetic particles may be used, as mentioned above, in the isolation of nucleic acids, for example. As a further illustrative example, the apparatus may be used for an organic compound to undergo a chemical reaction (such as e.g. reduction or oxidation). A product obtained by such chemical reaction may be of different hydrophilic properties when compared to the compound used as a starting material. As an illustrative example, a starting material or a reactant used may be too hydrophilic to bind to a surface of for instance a probe or beads possessing a hydrophobic surface. The reaction product may however be hydrophobic enough to be attracted to such a surface. Particles equipped with a suitable hydrophobic surface may thus bind a respective reaction product without binding starting material or a reactant. The particles may further be equipped with a magnetic core, so that they can be used to recover such a reaction product. In some embodiments, the magnetic particles are coated with a substance that has an affinity for nucleic acids or organic compounds. Accordingly, when agitated by the probe using any one of the above-mentioned agitation methods, a uniform distribution of magnetic particle is achieved throughout the chamber. The magnetic particles bind to the desired nucleic acid or reaction product and remain distributed throughout the chamber.
Subsequently, the insertion of the magnetic probe into the chamber creates a magnetic field, which attracts and holds the magnetic particles (and nucleic acids or reaction products bound thereto) to the probe. Consequently, a large proportion of the desired nucleic acid or reaction product can been removed from the medium (test sample) and the remaining medium is no longer essential. Hence, with the probe finally creating an outlet in the penetrable sealing layer, the unwanted medium may be released via the outlet and to the absorption layer to be absorbed.
Should there be a need to further process a sample of the extracted nucleic acid or recovered reaction product, such as washing, for example, the device, as mentioned above, may be arranged such that the outlet is in fluid communication with the inlet of another device. Such an arrangement may be achieved if several devices are collinearly stacked, for example. Accordingly, the probe of the above illustrative example may be further inserted through the outlet of the sealing layer, past the absorption layer and into the chamber of the subsequent device to further process the extracted nucleic acid as required.
As described above, target matter may be attracted or collected, for example to a probe inserted via the inlet of the device. Typically the method of the invention also includes a penetration of a sealing layer of the device (cf. e.g.
Further processing may include, but is not limited to washing, purification and removal of a product from magnetic particles, which is derived by means of a process carried out in the device, for example, a nucleic acid or the product of a chemical reaction. As an illustrative example, an extracted nucleic acid may be released from the probe or any capturing surfaces at the end of the process, into an elution chamber. The elution chamber may be provided as part of the apparatus.
In some embodiments it may be desired to further react a resulting organic compound, which is the product of a chemical reaction performed in a device of the invention. The chamber of the subsequent device may thus be used to perform such a further reaction process. Thus a series of steps of an organic synthesis may be performed using an arrangement of several devices of the invention.
As indicated above, the chamber of the device may be filled with a medium for carrying out a chemical or biological process. Examples of the media that may be used include, but are not limited to, organic solvents and buffer solutions. Examples of buffer solutions include, but are not limited to, lysis buffers, binding buffers and washing buffers such as phosphate buffered saline (PBS), for example.
Numerous organic solvents are used in the art and can be used to carry out the various processes described herein. Examples of solvents include, but are not limited to, hexane, heptane, cyclohexane, benzene, toluene, p-xylene, pyridine, dichloromethane, chloroform, carbon tetrachloride, carbon disulfide, tetrahydrofuran, dioxane, diethyl ether, diisopropylether, ethylene glycol monobutyl ether, tetrahydrofuran, methyl ethyl ketone, methyl isobutyl ketone, acetone, cyclohexanone, ethyl acetate, isobutyl isobutyrate, ethylene glycol diacetate, dimethylformamide, acetonitrile, N,N-dimethyl acetamide, nitromethane, acetonitrile, N-formylpiperidine, N-methylpyrrolidone, dimethylsulfoxide, methanol, ethanol, butyl alcohol, formic acid, dimethylarsinic acid [(CH3)2AsO(OH)], N,N-dimethyl-formamide, N,N-diisopropylethylamine, chlorophenol, acetic acid, tert.-butyl alcohol, phenol, cyclohexanol, and aniline, to name a few.
Numerous buffer compounds are used in the art and may be used to carry out the various processes described herein. Examples of buffers include, but are not limited to, solutions of salts of phosphate, carbonate, succinate, carbonate, citrate, acetate, formate, barbiturate, oxalate, lactate, phthalate, maleate, cacodylate, borate, N-(2-acetamido)-2-amino-ethanesulfonate (also called (ACES), N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (also called HEPES), 4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (also called HEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also called PIPES), (2-[Tris(hydroxymethyl)-methylamino]-1-ethansulfonic acid (also called TES), 2-cyclohexylamino-ethansulfonic acid (also called CHES) and N-(2-acetamido)-iminodiacetate (also called ADA). Any counter ion may be used in these salts; ammonium, sodium, and potassium may serve as illustrative examples. Further examples of buffers include, but are not limited to, triethanolamine, diethanolamine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris(hydroxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)methane (also called BIS-TRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (also called TRICINE), to name a few. The buffers may be aqueous solutions of such buffer compounds or solutions in a suitable polar organic solvent.
In addition to the above, the medium may also contain additives, such as, but not limited to, reagents, catalysts and reactants, for carrying out a chemical or biological process. As an illustrative example, salts, substrates or detergents may be added in order to maintain cells or proteins in an intact state. As a further illustrative example, chelating compounds may be required, for instance to protect organisms from traces of otherwise toxic salts or to increase the yield of a chemical reaction. As yet a further illustrative example, protease inhibitors may need to be added in order to maintain proteins in an intact state. A further example of a possible additive to a medium includes magnetically attractable particles (see above).
As an illustrative, but not limiting example, the device of the present invention may be used to carry out a sandwich-type enzyme-linked immunosorbent (ELISA) assay. The uses and capabilities of this assay are well known to those skilled in the art. By combining several devices, wherein each device contains at least one or more of the requisite reagents for the binding, washing and detection steps, it is possible to assay for the presence of target matter in a sample. As described above, the capture reagent may be directly or indirectly coupled to the probe. In a specific embodiment, the capture reagent is an antibody targeted to an antigen of interest. More specifically, the antibody may be directed to an antigen present in a HIV virus and the sample blood or serum suspected to contain this HIV virus. Detection of an antigen can be monitored through colorimetric and or spectroscopic analysis. This can occur after elution of substrates captured onto the probe or within the device itself without recourse to an elution step.
The following drawings, accompanying descriptions thereof, and the subsequent examples serve in illustrating the invention and to further aid in the clarity and understanding of the invention. However, it should be noted that the scope of the invention is by no means limited to the various embodiments illustrated hereafter.
A close-up view of these depicted stages, focussing on the movement of the probe, is shown in
A close-up view of some of these embodiments is shown in
In a standard device for processing a sample, a sample is introduced into a container 1 (
In contrast to this prior art, the device of the present invention does not require removing the sample from a container. As shown in
As schematically depicted in
Between the lower chamber on the left and the chamber on the right side of this chamber (into which a probe 13 is inserted) there is a sealing layer 9. This sealing layer can form an opening in a reversible manner. Thus the sealing layer may then be opened. Pressure may be applied or the device may be rotated clockwise (around the axis defined by the respective sealing layer), so that fluid 10 together with target matter 54 is moved into the depicted upper sample processing chamber 2 on the right, as shown in step II (lower part of
The present example illustrates the manufacture of an exemplary device of the present invention. The exemplary device contains five chambers, of which four are designed for usage as sample processing chambers and one to house an elution chamber.
A 32.0 mm×32.0 mm square transparent acrylic block (Perspex) with the thickness of 20.0 mm was selected to fabricate the processing chamber. The shape of the sample processing chamber was formed by CO2 laser drilling, such that a hole of 15.0 mm diameter was generated in the center of the square block. This hole formed part of the interior of the sample processing chamber (to be completed by the sealing layer) and its diameter was determined to have a capacity of more than 2.0 ml volume. The inlet of the sample processing chamber was formed on the second large side of the block, opposing the side where the hole had been formed. This inlet had the form of a second hole of a diameter of 2.0 mm. This inlet thus allows the injection of fluid and other matter required for processing. Alignment holes of 3.0 mm diameter were also laser drilled on the four corners of block (i.e. not contacting the chamber), when seen from the top of one of the two large sides, for example from the inlet. These alignment holes later facilitated accurate alignment during assembly.
A penetrable plastic sealing layer was made of acrylic material obtained from 3M with a thickness of 0.025 mm to 0.05 mm. The quality of this material is similar to those materials used in projector transparencies. The lateral dimension of the penetrable plastic sealing corresponded to the respective dimension of the preformed sample processing chamber (15.0 mm diameter, see above), so that the sealing layer would complete the chamber. A perforation line with a cross-shape as shown in
A 32.0 mm×32.0 mm square transparent acrylic block (Perspex) with a thickness of 4.0 mm was selected for the supportive layer. A recess was fabricated in the supportive layer, so that the recess is able to accommodate the absorption layer. Hence, the supportive layer forms a housing for the absorption layer. The recess was again created by means of CO2 laser drilling. An internal square shape was excised out of the block thus forming the recess to house the absorbing material. Thus one side of the housing was open and was later (see below) completed to form a hollow space by placing the sealing layer on top of it. A 3.0 mm diameter alignment hole was also CO2 laser drilled on each of the four corners of block, when seen from the top of one of the two large sides, with the same alignment coordinates as the processing chamber.
The penetrable absorption layer was made of nitrocellulose material that was obtained from Whatman. Two sublayers of the nitrocellulose material were prepared, the first one 2.0 mm thick, and the second one 0.2 mm thick. The lateral dimension of both sublayers was cut to the same dimension as the inner dimension of the housing described in (c), in order to allow the two sublayers to fit exactly into the housing. A hole with a diameter corresponding to the sealing layer (and thus the sample processing chamber) was formed in the first subabsorption layer of 2.0 mm thickness. A perforation line with a cross-shape as shown in
A chamber resembling the sample processing chamber was manufactured as described above. A 32.0 mm×32.0 mm square aluminium block with a thickness of 8 mm was prepared. This block was used to seal the opening of 15.0 mm diameter (see above) instead of the sealing layer used above. Aluminium material was selected to afford heating and cooling at a faster rate without mechanical deformation. The chamber contains an extruded funnel shape chamber wherein the elution chamber can be placed (cf. e.g.
A 5.0 mm thick stainless steel fixture with dimension of 100.0 mm×50.0 mm was used as a base for the assembly. A long stainless steel rod with the diameter of 2.0 mm and a length of 200.0 mm was mounted on the four corners of the fixture. The rods served as the skeletal frame of the device. Hence, the mounting location of the rods is constrained to exactly align with the four corner holes of the sample processing chambers, penetrable sealing layers, and supportive layers (the absorption layer is smaller with respect to its width and does not contain alignment holes).
The assembly starts with mounting onto the stainless steel fixture the chamber that serves to house the elution chamber. The top opening of this chamber was then covered by a penetrable sealing layer using a silicon glue.
A housing for the absorption layer (see above) was mounted on top of the chamber with the sealing layer. The two sublayers of the absorption layer were inserted separately. The 2.0 mm thick subabsorbtion layer was inserted first. Thereafter the subabsorbtion layer of 0.2 mm was placed on top of the subabsorbtion layer of 2.0 mm.
The penetrable sealing layer completes both the housing of the absorption layer, i.e. turns the supportive layer into a housing, and completes the sample processing chamber. It can thus principally be attached to either element first. For the assembly of the device, a sealing layer was used to complete the sample processing chamber in a first step. For this purpose two sealing layers were used for each chamber. One sealing layer was applied to the remaining opening of the sample processing chamber as described above. A second sealing layer was applied to the inlet of the sample processing chamber. The sealing layers were fused to the chamber using silicon glue. With the penetrable sealing layer already fused thereto, the processing chamber was mounted on top of the absorption layer. Hence, the housing of the absorption layer was completed in this step. In this configuration, the sample processing was on top of the absorption layer, which was located on top of the supportive layer (forming a housing for the absorption layer), and the supportive layer was on top of the chamber that serves to house the elution chamber, as for instance depicted in
The cycle of layering a supportive layer, an absorption layer, and a sample processing chamber with a sealing layer at its lower end was repeated three times. The device was ready to be used for four processing steps and one elution step.
The present example illustrates the manufacture of an exemplary probe in the manufacture of an exemplary apparatus of the present invention.
The hollow primary core rod (outer sheath) of the probe was formed as a cylindrical tube, and made up of high density polyethylene material. The length of the tube was about 150 mm with an outer diameter of 4.0 mm and an internal diameter of 2.5 mm. The head of the probe, which can accommodate an internal magnetic core, together with the arms was fabricated in a separate step as an end-part by injection moulding. The hollow primary core rod and the end-part were joined by glue.
A cylindrical neodymium magnet with the diameter of 2.0 mm and a length of 5.0 mm was mounted on the tip of a 2.0 mm stainless steel rod using glue. The rod was approximately 200.0 mm long.
The present example illustrates the combination of an exemplary agitation motor and a device of the present invention (as described above) in the manufacture of an exemplary apparatus of the present invention. In this embodiment a motor and a vertical guide are part of the apparatus of the invention.
The probe (cf. Example 2) was inserted into the device that had been manufactured as described in Example 1. A probe holder was selected to be able to match the outer diameter of the probe. The probe holder was a hollow cylindrical device with bearings for rotation. The probe holder was used for attaching and removing the hollow primary core rod of the probe in a manner as depicted in
The base plate with the motor and the probe holder was fixed to a vertical guide. The vertical guide was made up of lead screw (cf.
The present example illustrates an exemplary application of the method of the present invention. An apparatus of the invention, manufactured and assembled as described above, and including five sample processing chambers and one chamber to house an elution chamber, was used to extract Dengue virus RNA. A conventional microtube, pre-loaded with 50 μl of sterile deionised water, had been placed into the chamber to house the elution chamber (cf. Example 1e).
The acrylic and plastic sealing materials used in this experiment were treated overnight with 0.1 M HCl and later treated with RNase Zap buffer (Ambion Diagnostics, Austin, Tex., USA) was used to prevent contamination. The extraction of Dengue RNA was performed in three independent identical experiments.
The first of the five sample processing chambers was preloaded with 250 μl of lysis buffer and 750 ml of binding buffer. A silica coated magnetic beads with the diameter of 5 um of was introduced into the mixture. A volume of 1.0 ml of 50% ethanol with water was introduced to the sample processing chambers, which were located below the first sample processing chamber, for washing steps. For this experiment, four processing chambers for four washing steps were prepared. A volume of 50 μl of deionised water had already been introduced into an elution chamber that was located in a fifth chamber.
A 50 μl whole blood sample was prepared, and was spiked with 15,000 pfu of inactivated Dengue virus. The blood sample was injected to the first processing chamber for 15 minutes for effective lysis of the cells and binding of the nucleic acids to the silica coated magnetic beads. The plastic probe (as described in the fabrication examples), which was already situated inside the processing chamber, without the inner magnetic core on the tip was used to agitate the mixture. After the processing time was completed, the core of the probe with a magnetic end was lowered into a collection position as illustrated in
The probe with the magnetic beads bound to it penetrated the sealing layer, thus establishing fluid contact of the sample processing chamber with the absorption layer. The absorption layer absorbed the supernatant released from the sample processing chamber. Penetration of the sealing layer and absorption of the supernatant occurred almost simultaneously. The penetration was controlled by the vertical guide (as describe in fabrication example 3). The probe was securely held in the required level in the next sample processing chamber. In this sample processing chamber a washing step was performed. The inner magnetic core of the probe was retracted up by 50 mm, thereby allowing the release of magnetic beads in the washing buffer for 3 minutes, before the inner probe was lowered again into a collective position (cf.
In the lowest chamber, which served as a housing for the elution chamber, the probe, which had the magnetic beads bound to it, entered the elution chamber, which had been pre-loaded with 50 μl of deionised water. Heating was conducted by means of the hotplate located below the elution chamber (cf.
The eluent containing the RNA was characterized using Roche's Light Cycler, which is a Real-Time PCR Machine. A 10.0 μl volume of PCR mix was prepared with the set up reaction comprising 1× Taq buffer (Invitrogen), 0.2 mM dNTP, 4 mM MgCl2, 0.5 mg/ml BSA, 0.2× Syber Green I, 1.25 U Platinum Taq polymerase (Invitrogen), and 1.94 μl of eluent (isolated by the method of the present invention). Autoclaved deionised water served as a negative control.
Real time detection provided an amplification plot depicting the fluorescence signal versus reaction time expressed as cycle numbers (see
As can be seen from the above data and
The above illustrations and accompanying description of the various embodiments of the present invention merely serve to aid in the understanding principle of the said invention. Accordingly, it should not be construed that the present invention described herein is simply limited to the illustrated embodiments of the device and/or apparatus.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SG05/00241 | 7/19/2005 | WO | 00 | 9/26/2008 |
