Patent Application
20240316551
The present invention relates to a microfluidic circuit, microfluidic chip, kit and method for isolating and purifying an analyte, preferably a nucleic acid, according to the independent claims.
The field of analyzing nucleic acids using enzymatic reactions, such as in a polymerase chain reaction assay, generally requires the use of reaction enzymes and/or proteins which are very sensitive to reagents commonly used during the preceding nucleic acid isolation step, in particular isolation of DeoxyriboNucleic Acid (DNA) and RiboNucleic Acid (RNA).
The reagents commonly used in the isolation step facilitate lysis of the biological samples including biologic cells and/or virus material. In other words, the reagents facilitate that the outer boundary or cell membrane/virus wall is at least in part broken down or destroyed in order to release intra-cellular/intra-viral material, such as DNA, RNA, protein or organelles from a cell. Such a destruction requires suitable strong lysis agents, such as guanidine hydrochloride, alcohol, e.g., ethanol, isopropanol, or sodium dodecyl sulfate (SDS). The lysis agent may be used in form of a lysis buffer mixture, such as the SDS lysis buffer, which may comprise 0.5% (w/v) SDS, 0.05 M Tris·Cl, Adjust pH to 8.0 and add 1 mM fresh dithiothreitol (DTT).
Due to the strong lysis effect on peptidic structures, these lysis agents may, however, interfere with enzymes to be used for the analyzing step of nucleic acids, such as required for the polymerase chain reaction (PCR). Thus, there is a need for providing devices, which reduce the remaining lysis agent contamination prior to conducting the analyzing step.
In addition to lysis agents, washing agents used to wash and purify the isolated nucleic acid from the remaining cellular/wall fragments may also interfere with the enzymatic reaction on nucleic acids, such as PCR, in particular in case the washing agent is selected from a suitable alcohol, such as ethanol or isopropanol.
Generally, the lysate comprising the destroyed cells/virus, isolated nucleic acids and lysis agent is contacted with capturing agents, such as beads, preferably micro beads, in order to bind the nucleic acid to the beads and to, thus, isolate the bound nucleic acid beads from the destroyed cells/virus and further released inter-cellular/inter-viral material.
In case the enzymatic reaction to analyze the nucleic acid is performed on a microfluidic device, such as a microfluidic chip, the lysis agent and the washing agent have to be separated from the isolated nucleic acid, which is usually conducted by transferring them to a waste chamber, which also can be an outlet of the microfluidic device, whereas the isolated and eluted nucleic acid has to be transferred to a reaction chamber, where the enzymatic analyzing reaction takes place.
To facilitate the separation, the microfluidic chip generally comprises a channel system including at least one junction at which the channel branches into a sub-channel connecting in fluidic flow down-stream direction to the waste chamber and a sub-channel connecting in fluidic flow down-stream direction to the reaction chamber. The channel system comprises suitable means, such as two-state valves, to facilitate fluidic flow in direction to the waste chamber (WC) or to facilitate fluidic flow in direction to the reaction chamber (RC). As these valves cannot be arranged directly on the junction, at least some lysate, comprising the lysis agent and/or washing agent (LB), which is to be transferred in flow direction downstream (fd) to the waste chamber (WC), also enters at least part (distance d) of the sub-channel connecting to the reaction chamber (RC), which is illustrated in
Thus, there exists an ongoing need to increase analytical precision of enzymatic reactions on analytes of biologic samples, preferably nucleic acids, such as PCR.
Furthermore, there exists an ongoing need in providing simple and cost effective isolation and purification processes of analytes of biologic samples, preferably nucleic acids, which reduce or prevent remaining contamination, in particular with lysis agents, in the following enzymatic reactions.
Thus, it is an aim of the present invention to provide an alternative and/or improved microfluidic device, such as microfluidic circuit or microfluidic chip for isolating and purifying analytes, preferably nucleic acids from biologic samples providing a suitable analytical precision and/or reducing or preventing contamination with contamination agents, such as lysis agents and/or washing agents in the enzymatic analyzing reactions on the analytes, preferably nucleic acids, more preferably wherein the isolation and purification processes are simple and cost effective.
One or more problems of the present invention is/are solved by the subject matter of the independent claims, namely a microfluidic circuit/a microfluidic chip/a kit/a system for isolating and purifying analytes, preferably nucleic acids from biologic samples, and uses thereof, as well as a process for controlling fluidic flow in an isolation and purification procedure, and a process for isolating and purifying an analyte, preferably a nucleic acid, from a biological cell sample. Advantages (preferred embodiments) are set out in the detailed description hereinafter and/or the accompanying figures as well as in the dependent claims.
Accordingly, a first aspect of the present invention relates to a microfluidic circuit for use in a microfluidic chip for isolating and purifying an analyte, preferably a nucleic acid, comprised in a biologic sample, wherein the microfluidic circuit is arranged in a substrate and comprises a first microfluidic channel (MC1) fluidicly connecting an outlet of a buffer chamber (BC) with an inlet of an elution chamber (EC) and a second microfluidic channel (MC2) fluidicly connecting an outlet of the elution chamber (EC) with an inlet of a waste chamber (WC) and with a or connectable to a reaction chamber (RC), wherein the second microfluidic channel (MC2) is branched at a first junction (J1) into two downstream sub-channels (MC21, MC22) of a first generation, wherein the first sub-channel (MC21) connects to the inlet of the waste chamber (WC) and the second sub-channel (MC22) connects or is connectable to the reaction chamber (RC), characterized in that the second sub-channel (MC22) of first generation is branched at a second junction (J2) into two downstream sub-channels of second generation, wherein a first sub-channel (MC221) connects or is connectable to the reaction chamber (RC) and a second sub-channel (MC222) is connected to the buffer chamber (BC) and is configured to facilitate fluidic flow in at least part of the second sub-channel of first generation (MC22) and the second microfluidic channel (MC2) in direction to the outlet of the elution chamber (EC), wherein the fluidic flow direction is controlled by an arrangement of three or more fluidic flow control means, wherein the fluidic flow control means are independently actuatable to facilitate fluidic flow in a microfluidic channel (on-status) and/or actuatable to stop fluidic flow in a microfluidic channel (off-status).
According to a second aspect of the present invention, a microfluidic chip for isolating and purifying an analyte, preferably a nucleic acid, comprised in a biologic sample is provided having a sample chamber (SC) fluidicly connected with an inlet of a capturing chamber (CC), characterized in that an outlet of the capturing chamber (CC) is fluidicly connected to the microfluidic circuit according to first aspect of the present invention, wherein the outlet of the capturing chamber (CC) is connected with a third microfluidic channel (MC3) to a fourth junction (J4) of the first microfluidic channel (MC1).
According to a third aspect of the present invention, a kit is provided comprising or consisting of the microfluidic chip according to the second aspect of the present invention, and one or more, the same or different, suitable reaction agents selected from the group consisting of a reagent suitable for performing lysis of biologic sample (lysis agent); a reagent suitable for performing washing of lysed biologic cell fragments from the isolated analyte, preferably nucleic acid, respectively bound to at least part of the capturing carriers (washing agent); a reagent suitable for eluting the isolated analyte, preferably nucleic acid, from the capturing carriers (elution agent); a reagent suitable for suspending the sample (dissolution agent).
According to a fourth aspect of the present invention, a system configured to perform isolation and purification of an analyte, preferably a nucleic acid, from a biologic sample is provided, characterized in that it comprises or consists of a microfluidic chip according to the second aspect of the present invention and a thermocycler.
According to a fifth aspect of the present invention, a process for isolating and purifying an analyte, preferably a nucleic acid, from a biological cell sample comprising or consisting of the following steps is provided:
According to a fifth aspect of the present invention, a use of a microfluidic circuit according to the first aspect of the present invention or a microfluidic chip according to the second aspect of the present invention or a kit according to the third aspect of the present invention or a system according to the fourth aspect of the present invention for isolating and purifying an analyte, preferably a nucleic acid, from a biological sample is provided, preferably for conducting a biochemical assay selected from the group consisting of an enzymatic analysis of analytes of biological samples, preferably nucleic acids including DNA or RNA analysis, such as a polymerase chain reaction (PCR) and in particular a PCR with high-throughput sequencing.
The inventive aspects of the present invention as disclosed hereinbefore can comprise any possible (sub-)combination of the preferred inventive embodiments as set out in the dependent claims or as disclosed in the following detailed description and/or in the accompanying figures, provided the resulting combination of features is reasonable to a person skilled in the art.
Further characteristics and advantages of the present invention will ensue from the accompanying drawings, wherein
As set out in more detail hereinafter, the inventors of the different aspects of the present invention have found out that the inventive microfluidic circuit facilitates removing all residual contaminants, such as lysate, comprising lysis agent, and washing buffer, that could otherwise inhibit further reaction of the isolated analyte, preferably nucleic acid, in particular in an enzymatic analyzing reaction, such as polymerase chain reaction (PCR).
Furthermore, the use of magnetic capturing carriers for binding to the nucleic acid, isolated from the lysed biologic sample is advantageous, as the isolated analyte, preferably nucleic acid, respectively bound the magnetic capturing carriers, such as magnetic (micro) beads, can be located upon magnetic field excitation in the area of the eluting chamber (EC), preferably at least at part of the wall of the eluting chamber (EC).
Generally a magnetic field is used to agglomerate the analyte, preferably nucleic acid, bound to the magnetic capturing carriers in such a way that they form a magnetic conglomerate. This means, that the analyte, preferably nucleic acid, is predominantly integrated into the magnetic conglomerate so that any contamination agents, such as lysis and/or washing agents or elution agents are substantially hindered to integrate within the agglomerated analyte, preferably nucleic acid, bound conglomerate. In other words, the excitation of the magnetic field, which is generally unwanted due to its agglomeration effects, surprisingly reduces, preferably prevents unwanted analyte, preferably nucleic acid, elution from the magnetic capturing carriers during washing procedures. Thus, the precision of the analytical reaction, preferably analytical nucleic acid reaction, is increased by use of the magnetic capturing carriers. According to the present invention, the inventors could show that only about 5 wt. % of the analyte, in particular nucleic acid, respectively bound to the magnetic capturing carriers are washed to the waste chamber. This reduced amount of analyte, preferably nucleic acid, loss is in particular advantageous as all residual lysing and washing agents, which may act as inhibitors to the further enzymatic procedure on the analyte, preferably nucleic acids, are at the same time also removed to the waste chamber.
In order to elute the analyte, preferably nucleic acid, respectively bound to the magnetic capture carriers, the present invention teaches to excite the agglomerated analyte, preferably nucleic acid, bound capture carrier conglomerate with a suitable ultrasound impulse. This suitable ultrasound impulse destroys the agglomerated analyte, preferably nucleic acid, bound magnetic conglomerate and results in separated magnetic capture carriers respectively bound with analyte, preferably nucleic acid, suspended in the suitable elution agent, wherein the elution agent can efficiently elute the analyte, preferably nucleic acid, from the magnetic capture carriers.
In accordance with the present invention the term “analyte” includes suitable analytes to be isolated from biologic sample, preferably peptidic analytes or nucleic acid analytes or bacterial or viral analytes. Preferably, the analyte according to the present invention is regarded a nucleic acid analyte to be isolated from a biologic sample. Furthermore, in case the present invention uses the term “nucleic acid”, it also refers to any other kind of suitable analyte to be isolated and purified from a biologic sample unless otherwise indicated.
The expression “microfluidic circuit” means according to the present invention that a microfluidic channel system with suitable chambers is arranged in a suitable substrate. At least the microfluidic channels and the elution chamber of the inventive microfluidic circuit are configured to facilitate microfluidic flow properties. In particular, the microfluidic flow properties include that aqueous fluids to not mix with oleic fluids.
The expression “fluidicly connecting [ . . . ] with a or connectable to a reaction chamber” or “connected or connectable to a reaction chamber” or “connects or connectable to a reaction chamber” means according to the present invention that the second microfluidic channel may connect to a reaction chamber, which is already present in the substrate. It may connect directly to the reaction chamber or one or more (reservoir) chambers may be interposed. Alternatively, the second microfluidic channel may connect to an outlet of the microfluidic circuit according to the present invention, wherein the outlet is connectable directly to a reaction chamber (RC) or is connectable to a reaction chamber (RC) interposed with one or more other chambers.
According to the first aspect of the present invention, the inventive microfluidic circuit for use in a microfluidic chip for isolating and purifying an analyte, preferably a nucleic acid, comprised in a biologic sample, is arranged in a substrate and comprises a first microfluidic channel (MC1) fluidicly connecting an outlet of a buffer chamber (BC) with an inlet of an elution chamber (EC). It further comprises a second microfluidic channel (MC2) fluidicly connecting an outlet of the elution chamber (EC) with an inlet of a waste chamber (WC) and with a or connectable to a reaction chamber (RC). The second microfluidic channel (MC2) is branched at a first junction (J1) into two downstream sub-channels (MC21, MC22) of a first generation. The first sub-channel (MC21) connects to the inlet of the waste chamber (WC) and the second sub-channel (MC22) connects or is connectable to the reaction chamber (RC). According to the present invention the second sub-channel (MC22) of first generation is branched at a second junction (J2) into two downstream sub-channels of second generation, wherein a first sub-channel (MC221) connects or is connectable to the reaction chamber (RC) and a second sub-channel (MC222) is connected to the buffer chamber (BC) and is configured to facilitate fluidic flow in at least part of the second sub-channel of first generation (MC22) and the second microfluidic channel (MC2) in direction to the outlet of the elution chamber (EC). According to the present invention the fluidic flow direction is controlled by an arrangement of three or more fluidic flow control means, wherein the fluidic flow control means are independently actuatable to facilitate fluidic flow in a microfluidic channel (on-status) and/or actuatable to stop fluidic flow in a microfluidic channel (off-status). According to the present invention the buffer chamber (BC) connected or connectable to the elution chamber (EC) may be comprised in the inventive microfluidic circuit. Preferably the buffer chamber (BC) comprises in use an elution buffer. According to the present invention, the buffer chamber (BC) is preferably configured to pass the fluid in one flow direction. Such a configuration may include suitable syringes, which may be actuatable by pressure or electronic means.
Thus, in view of the second junction of the second microfluidic channel, the inventive microfluidic circuit is adapted to facilitate bidirectional fluidic flow at least between the outlet of the elution chamber (EC) to the second junction (J2), i.e. downstream of the elution chamber (EC). In view of this bidirectional fluidic flow properties, the inventive microfluidic circuit facilitates to remove any residual contaminant, which may impair the enzymatic reaction of the nucleic acids in the reaction chamber. Furthermore, the inventive microfluidic circuit facilitates the isolation and purification reaction by use of one waste chamber. The waste chamber may be in form of a void in a substrate, it may also effect removal of constituents from the microfluidic system by representing a waste reservoir of any shape connected to the substrate by an outlet of the substrate (e.g., the shape of the waste reservoir may be container, channel, chamber, etc. or alternatively the environment may be the waste reservoir). In other words, according to the first alternative the waste reservoir is part of the substrate of the microfluidic circuit, whereas according to the second alternative the waste reservoir of any shape is at least in operation connected to the microfluidic circuit by an outlet of the substrate of the microfluidic circuit.
In view of the present invention, the respective microfluidic circuits, its channels, the elution chamber, the waste chamber, the reaction chamber and the capturing chamber are generally configured as suitable voids or recesses in a suitable substrate. The material of a suitable substrate may be liquid and/or gas, preferably liquid and gas impermeable. As a counter example, polydimethylsiloxane (PDMS) is not regarded a fluid and gas impermeable substrate, as the substrate will be gas permeable. Furthermore, the substrate material may preferably not interfere with the processing of the fluid. In addition or alternatively, the material of the substrate may facilitate in particular thermal and optical processing of the liquid, such as irradiation with electromagnetic waves, such as in the infrared and/or ultraviolet range and/or visible range and or optical inspection. Accordingly, the material of the basic substrate is preferably transparent to the respective irradiated and/or emitted wavelength, in order to reduce a negative impact on processing of the aliquot of fluid. In addition or alternatively, the material of the substrate may not interfere with magnetic field and ultrasound impulses. Thus, according to an additional or alternative embodiment of the present invention, the substrate material may be selected from the group consisting of suitable glass and suitable polymers, wherein the polymers are preferably selected from the group consisting of polycarbonate, cyclic olefin copolymer, polystyrene, cyclic olefin polymer or poly(methyl methacrylate). More preferably, the substrate material is selected from polycarbonate.
According to an alternative or additive embodiment of the first inventive aspect, the first microfluidic channel (MC1) is branched at a third junction (J3) into two sub-channels of first generation. A first sub-channel (MC11) connects to the inlet of the elution chamber (EC) and a second sub-channel (MC12) connects to the second sub-channel (MC222) of second generation of the second microfluidic channel (MC2) thereby bypassing the elution chamber (EC). According to this embodiment, the same buffer chamber can be used for the bidirectional flow.
According to an alternative or additive embodiment of the first inventive aspect, the first and second microfluidic channels (MC1, MC2) and their respective sub-channels as well as the elution chamber (EC) are configured to facilitate microfluidic flow properties including prevention of mixture of oil and aqueous fluids, preferably are configured to respectively have an inner diameter perpendicular to the flow direction of 2 mm or less. As an example the inner diameter of a microfluidic channel may be 0.8 mm to 0.5 mm or less and the inner diameter of the elution chamber may be 0.5 mm to 2.0 mm or less. Such a configuration facilitates the use of a liquid oil phase, in particular of the elution buffer in order to further increase the removal of aqueous contamination agents, such as lysis agents and/or washing agents.
According to an alternative or additive embodiment of the first inventive aspect, the buffer chamber (BC) is configured as a syringe adapted to operate in one fluidic flow direction. The fluidic flow direction is generally towards the inlet of the elution chamber (EC). In the case the first microfluidic channel bypasses the elution chamber (EC) and connects to the second sub-channel of the second generation of the second microfluidic channel, then the fluidic flow direction may also follow this channel connection towards the outlet of the elution chamber (EC).
According to the first inventive aspect, the fluidic flow direction is controlled by an arrangement of three or more fluidic flow control means. These fluidic flow control means are generally independently actuatable to facilitate (unimpeded) fluidic flow in a microfluidic channel (on-status) and/or actuatable to stop/prevent fluidic flow in a microfluidic channel (off-status). This means, that the fluidic flow control means according to the present invention can exhibit at least two states, namely the on-status and the off-status (facilitated by so called multi state valves or two state valves), and optionally may also exhibit one or more intermediate valve states, where the fluidic flow is not completely, but partially blocked or opened (facilitated by multi state valves). Accordingly, the fluidic flow control means may refer to multi-state valves, and in particular to two-state valves in order to control the fluidic flow according to the present invention. An example of a multi-state valve is disclosed in U.S. Pat. No. 7,992,591 B2 (filed on Dec. 6, 2008), wherein microfluidic magnetic valves switch between “on-status” and “off-status” (also called “closed state”) and furthermore exhibit a partially blocked “constricting” state. For the present invention it is, thus, only relevant that the fluidic control means are switchable between the on-status and the off-status, so that any multi-state valve and in particular two-state valve, independently of the transition time between the on-state and the off-state is suitable as fluidic flow control means for the present invention. The fluidic flow control means may be actuatable by electric means, pressure means or any other suitable means, such as magnetic forces or equivalent forces. Particularly preferred valves are disclosed in the European patent application 19 186 118.6 (also published as EP 3 763 439 A1). The respective disclosure concerning the microfluidic valve disclosed in European patent application EP 3 763 439 A1 is incorporated herein by reference.
According to the present invention, the fluidic flow control means, preferably multi-state valves, in particular two-state valves (also called on/off valves) are used for routing fluid to different locations of the inventive microfluidic circuit.
According to an alternative or additive embodiment of the first inventive aspect, a fluidic flow control means (V1) is arranged to control fluidic flow in the first sub-channel (MC21) of first generation of the second microfluidic channel (MC2) connecting to the inlet of the waste chamber (WC), a further fluidic flow control means (V2) is arranged to control fluidic flow in the first sub-channel (MC221) of second generation of the second microfluidic channel (MC2) connecting or connectable to the reaction chamber (RC), and a further fluidic flow control means (V3) is arranged to control fluidic flow in the second sub-channel (MC222) of second generation of the second microfluidic channel (MC2). According to an alternative or additive embodiment, the multi-state valve, in particular the two-state valve is preferably used as fluidic flow control means.
According to an alternative or additive embodiment of the first inventive aspect, the waste chamber (WC) represents either a void in the substrate or represents a waste reservoir of any shape connected to the substrate by an outlet of the substrate.
All features and embodiments disclosed with respect to the first aspect of the present invention are combinable alone or in (sub-)combination with each of the further aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a person skilled in the art.
According to the second aspect of the present invention, the inventive microfluidic chip for isolating and purifying an analyte, preferably a nucleic acid, comprised in a biologic sample has a substrate comprising sample chamber (SC) fluidicly connected with an inlet of a capturing chamber (CC), characterized in that an outlet of the capturing chamber (CC) is fluidicly connected to the microfluidic circuit according to the first inventive aspect, wherein the outlet of the capturing chamber (CC) is connected with a third microfluidic channel (MC3) to a fourth junction (J4) of the first microfluidic channel (MC1). The fourth junction may be arranged between the buffer chamber (BC) and the third junction (J3) of the first microfluidic channel (MC1) or between the third junction (J3) of the first microfluidic channel (MC1) and the inlet of the elution chamber (EC).
According to an alternative or additive embodiment of the second inventive aspect, a fluidic flow control means (V4), preferably a multi-state valve, in particular a two-state valve, is arranged to control fluidic flow in the third microfluidic channel (MC3) between the outlet of the capturing chamber (CC) and the fourth junction (J4) of the first microfluidic channel (MC1). The fluidic flow direction is towards the inlet of the elution chamber (EC).
According to an alternative or additive embodiment of the second inventive aspect, the capturing chamber (CC) comprises suitable magnetic capturing carriers adapted to bind isolated analyte, preferably nucleic acid, lysed from a biologic sample. According to an alternative or additive embodiment of the second inventive aspect, the suitable magnetic capturing carriers are selected from suitable bead, preferably microbeads, optionally wherein the beads/microbeads have the same or differing dimensions. Suitable capturing carriers include magnetic silica beads or magnetic silica powder. In case of nucleic acids as respective analytes, the magnetic capturing carriers, in particular magnetic silica beads/powders are negatively charged and, therefore, easily bind the negatively charged nucleic acids (suitable preparations are available by Merck under the MagPrep® product line). The dimensions of the capturing carriers are generally adapted to facilitate transportation through the inventive microfluidic circuit. Preferably the diameter of suitable magnetic capturing carriers, in particular magnetic silica beads/microspheres/powder is in the range until 500 nm, preferably 50 to 400 nm, more preferably 100 to 200 nm.
According to an alternative or additive embodiment of the second inventive aspect, the capturing chamber (CC) is configured to have an inner volume to receive up to 5 ml, preferably between 1 to 4 ml, more preferably between 1.5 to 3 ml, in particular up to 2 ml of a fluid. In other words, the inner volume of the capturing chamber (CC) may exhibit in addition to the suitable amount of capturing carriers the respective volumes of fluid. Such a volume dimension facilitates a suitable binding of the analyte, preferably nucleic acid, with the capturing agents.
According to an alternative or additive embodiment of the second inventive aspect, the sample chamber (SC) is configured to receive an inner volume of up to 3 ml, preferably 0.5 ml to 2 ml, preferably up to 1 ml of a fluid. The sample chamber may also be configured to direct fluidic flow in one direction, preferably it may be configured as a syringe with fluidic flow in one direction.
According to an alternative or additive embodiment of the second inventive aspect, the microfluidic chip further comprises a lysis agent chamber (LC) and/or a dissolution agent chamber (DC) respectively in fluidic connection with the sample chamber (SC) and/or the capturing chamber (CC). In particular, the dissolution agent chamber (DC) may be fluidicly connected with the sample chamber (SC) and the lysis agent chamber (LC) may be fluidicly connected either with the sample chamber (SC) or with the capturing chamber (CC). Furthermore, the microfluidic chip may further comprise a washing agent chamber (WA) which may be in fluidic connection with the inlet of the elution chamber.
All features and embodiments disclosed with respect to the second aspect of the present invention are combinable alone or in (sub-)combination with each of the other aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a person skilled in the art.
According to the third aspect of the present invention, the inventive kit comprises or consists of the microfluidic chip according to second aspect of the present invention, and one or more, the same or different, suitable reaction agents selected from the group consisting of a reagent suitable for performing lysis of biologic sample (lysis agent); a reagent suitable for performing washing off lysed biologic cell fragments from the isolated analyte, preferably nucleic acid, respectively bound to at least part of the capturing carriers (washing agent); a reagent suitable for eluting the analyte, preferably isolated nucleic acid, from the capturing carriers (elution agent); a reagent suitable for suspending the sample (dissolution agent).
Generally the elution agent (synonym elution buffer) is suitable to elute the respective analyte, preferably nucleic acid, from the capturing carrier, e.g. buffer agents containing in particular Tris (tis(hydroxymethyl)aminomethane). In particular the buffer may be suitably adapted in its concentration and/or in its pH value with HCl to elute deoxyribo nucleic acids (DNA) or ribonucleic acids (RNA), wherein the RNA elution buffer may suitably exhibit a higher concentration of Tris and a higher pH value in comparison to DNA elution buffers. Suitable compositions are commercially available as Tris Base from Roth.
Generally, the lysis agent is suitable to lyse the biologic sample, such as cells, in particular human cells, bacterial cells or viral cells. Lysis agents suitable for bacterial cells may generally contain one or more constituents selected from the group consisting of salts, e.g. organic salts, such as Tris HCl, e.g. inorganic salts, such as NaCl; and detergents, e.g. anionic detergent SDS (sodium dodecyl sulfate/sulphate), non-ionic Triton X 100 with the following formula I, wherein n is 9 or 10 (MW: 647)
Alternatively, enzymatic lysis agents for bacterial lysis may contain one or more enzymes, such as selected from Mutanolysin, Trehalose (Proteinase A&A, Mutanolysin A&A, Trehalose Roth).
Furthermore, the lysis of bacterial cells may be conducted using a chemical mixture comprising a suitable alcohol including isopropanol, suitable salts and detergents.
Lysis agents suitable to destruct viral cells generally comprise Guanidinium thiocyanate and optionally buffer salts, such as Tris. The lysis agent may be adapted in view of the concentration of Guanidinium thiocyanate as well as of the pH value.
Suitable washing agents may generally comprise suitable buffer salts, such as Tris buffers. As with respect to the lysis agent, the washing agent may be adapted with respect to the respective analytes, in particular DNA or RNA with respect to the salt concentration and pH value. The washing agents may optionally contain further constituents such as reducing agents, e.g. dithiothreitol (DTT), in order to reduce disulfide bonds and to protect from oxidization.
All features and embodiments disclosed with respect to the third aspect of the present invention are combinable alone or in (sub-)combination with each of the other aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable.
According to the fourth aspect of the present invention, the inventive system configured to perform isolation and purification of an analyte, preferably nucleic acid, from a biologic sample, characterized in that it comprises or consists of a microfluidic chip according to second aspect of the present invention and a thermocycler.
The thermocycler (also known as a thermal cycler, PCR machine or DNA amplifier) is a device which comprises heating/cooling means, such as a thermal block or other heating/cooling means, to facilitate a polymerase chain reaction with nucleic acids, in particular the amplification of segments of nucleic acid, or other temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
According to an alternative or additive embodiment of the second inventive aspect, the system further comprises or consists of a magnetic field excitation means arranged in use in such a distance to the elution chamber (EC) of the microfluidic chip that upon magnetic field excitation at least part of the magnetic capturing carriers form an aggregated magnetic conglomerate comprising analyte, preferably nucleic acid, bound thereto, further preferably located due to the magnetic field at a wall of the elution chamber (EC). Generally, the means of magnetic field excitation may comprise a permanent magnet, such as a neodymium magnet, that preferably can be moved relative to the microfluidic channel. Alternatively, an electromagnet may be selected as magnetic excitation means. The distance of the magnetic excitation means to the inventive microfluidic circuit is suitably chosen to exhibit the magnetic effect within the microfluidic channel and in particular the elution chamber. Accordingly, the magnetic field excitation means is arranged suitably close to the inventive microfluidic circuit, preferably in a distance of less than 5 mm, more preferably less than 1 mm.
According to an alternative or additive embodiment of the second inventive aspect, the system further comprises an ultrasound excitation means arranged in use in such a distance to the elution chamber (EC) of the microfluidic chip that upon ultrasound impulse excitation the aggregated magnetic conglomerate is disaggregated at least in part into separate magnetic capturing carriers respectively with analyte, preferably nucleic acid, bound thereto so that the analyte, preferably nucleic acid, can be eluted from the magnetic capturing carriers. The means of ultrasound excitation may be chosen from a suitable ultrasonic transducer. The ultrasound excitation means is generally arranged in relation to the inventive microfluidic circuit in such a way that it may transmit in use respective acoustic waves through a suitable medium, such as a non-permeable substrate/membrane to the fluid located in use within the inventive microfluidic circuit, in particular the elution chamber thereof.
All features and embodiments disclosed with respect to the fourth aspect of the present invention are combinable alone or in (sub-)combination with each of the other aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable.
According to the fifth aspect of the present invention, the inventive process for isolating and purifying an analyte, preferably a nucleic acid, from a biological cell sample comprises or consists of the following steps:
All features and embodiments disclosed with respect to the fifth aspect of the present invention are combinable alone or in (sub-)combination with each of the other aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable.
According to the sixth aspect of the present invention, the inventive use of a microfluidic circuit according to the first inventive aspect or a microfluidic chip according to the second inventive aspect or a kit according to the third inventive aspect or a system according to the fourth inventive aspect for isolating and purifying an analyte, preferably a nucleic acid, from a biological sample, preferably for conducting a biochemical assay selected from the group consisting of an enzymatic analysis of nucleic acids including DNA or RNA analysis, such as a polymerase chain reaction (PCR) and in particular a PCR with high-throughput sequencing.
All features and embodiments disclosed with respect to the sixth aspect of the present invention are combinable alone or in (sub-)combination with each of the other aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a person skilled in the art.
Further characteristics and advantages of the present invention will ensue from the following description of embodiments of the inventive aspects with reference to the accompanying drawings.
As already discussed in the background part of the present invention,
In order to reduce the lysis buffer or any other contamination during the reaction procedure, the present invention provides the following solution.
According to the present invention the microfluidic channels, including the first and second microfluidic channels (MC1, MC2) and their respective sub-channels as well as the elution chamber are configured to facilitate microfluidic flow properties including prevention of mixture of oil and aqueous fluids. According to an additional or alternative embodiment, the microfluidic channels, including the first and second microfluidic channels (MC1, MC2) and their respective sub-channels as well as the elution chamber are configured to respectively have an inner diameter perpendicular to the flow direction of 2 mm or less. As an example the microfluidic channels have an inner diameter of 0.8 mm to 0.5 mm, whereas the elution chamber has an inner diameter of 0.5 mm to 2.0 mm.
The fluidic flow properties are in particular illustrated in
According to
According to
Then, as illustrated in
According to
Then the elution buffer (EB) is transferred from the buffer chamber 14 via the inlet of the elution chamber 15 (not illustrated) to the second microfluidic channel 12 and further through the first junction J1 to the waste chamber 16 so that the remaining lysis buffer is transferred through the sub-channel 121 to the waste chamber 16. Subsequently, the elution buffer is transferred through the first junction J1 and the sub-channel 122 through the junction J2 and the sub-channel 1221 to the reaction chamber 17. Accordingly, the eluted analyte, preferably nucleic acid, is preferably free of the remaining contaminants.
According to
According to
According to
All of the features disclosed with respect to the accompanying figures can alone or in any sub-combination be combined with features of the three aspects of the present invention including features of preferred embodiments thereof, provided the resulting feature combination is reasonable to a person skilled in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21150910.4 | Jan 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/050417 | 1/11/2022 | WO |
