Immobilizing a fluid sample for inhibiting spatial spreading

Abstract

The invention relates to immobilizing a fluid sample for inhibiting spatial spreading. A method of processing a fluid sample (100) in a sample separation device (10), wherein the method comprises: at least partially immobilizing the fluid sample (100) by a fixation agent (102), thereby inhibiting spatial spreading of the fluid sample (100); and subsequently at least partially releasing the fluid sample (100) from the fixation agent (102).

Description

TECHNICAL FIELD

[0001] The present invention relates to a method of handling a fluid sample in a sample separation device and to a sample separation device. BACKGROUND

[0002] In liquid chromatography, a fluid sample and eluent (liquid mobile phase) can be pumped through conduits and a separation unit, such as a column, in which separation of sample components takes place. The column can comprise a material capable of separating different components of the fluid sample. The separation unit can be connected through conduits to other fluidic members, such as a sampler or injector, a detector. Prior to the fluid sample being introduced into a separation path between a fluid driving unit, in particular a high pressure pump, and the separation unit, a predetermined amount of fluid sample should be introduced from a sample source, such as a sample container, via a syringe needle into a sample loop by a corresponding movement of a piston within a metering device. Thereafter, the injector valve is switched to introduce the introduced amount of fluid sample from the sample loop in the metering path into the separation path between the fluid driving unit and the separation unit for subsequent separation.

[0003] Another sample separation technique is gel electrophoresis. Gel electrophoresis is a method of separation and analysis based on the size and charge of a fluid sample containing, for example, macromolecules such as DNA and proteins and fragments thereof. Different fractions of the fluid sample, such as different nucleic acid molecules, are separated by applying an electric field to move the negatively charged molecules through a gel medium, such as agarose or other substances. Shorter molecules move faster and migrate further than longer molecules, because shorter molecules more easily migrate through the pores of the gel.

[0004] WO 2008 / 014825 discloses a fluidic device comprising a substrate, e.g. a glass substrate, and a transport medium, e.g. a gel, provided, e.g. printed, on said substrate to define a transport path for transporting a fluid sample, e.g. a biochemical liquid to be analyzed, driven by an external source, e.g. electrical power applied to electric contact pins to electrically connect to the transport medium in electrophoresis applications.

[0005] Gutzweiler et al. (“Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale”, 2017, Electrophoresis, vol. 38, pp. 1764-1770) disclose on-demand electrophoretic separation of DNA molecules in an open microfluidic system on a planar polymer substrate. The open microfluidic separation system comprises two opposing reservoirs, a semi-contacting writing gel line serving as a separation channel interconnecting the reservoirs, and a sample injected into the gel line via non-contact droplet dispensing.

[0006] However, spatial broadening of a fluid sample, for example within a waiting time prior to separation, can impair the spatial precision of sample separation in the above and other sample separation devices prior to and during separation. SUMMARY

[0007] It is an object of the present application to separate a sample with high precision. This object is achieved by the independent claims. Further embodiments are evident from the dependent claims.

[0008] According to one exemplary embodiment of the present application, a method of handling a fluid sample in a sample separation device is provided, wherein the method comprises at least partially immobilizing (in particular, inhibiting free movement of) the fluid sample by a fixation agent (in particular, by forming at least one bead comprising the fluid sample and the immobilized fixation agent), thereby inhibiting spatial broadening of the fluid sample; and subsequently at least partially releasing or removing the fluid sample from the fixation agent (in particular, the bead comprising the fixation agent) (in particular, restoring or reestablishing free mobility of the fluid sample).

[0009] According to another exemplary embodiment, a sample separation device for separating a fluid sample is provided, wherein the device comprises an immobilization unit configured to at least partially immobilize the fluid sample by a fixation agent, thereby inhibiting spatial broadening of the fluid sample.

[0010] In the context of the present application, the term "fluid sample" can in particular represent any liquid and / or gaseous medium, optionally further comprising solid particles to be analyzed. Such fluid sample can comprise a plurality of fractions of molecules or particles that shall be separated, e.g. small mass molecules or large mass biomolecules such as proteins. Separating a fluid sample into fractions involves a certain separation criterion according to which the separation takes place, such as charge, mass, volume, chemical property, etc. For example, such fluid sample can be a biological sample that can contain components to be separated, such as proteins and / or DNA.

[0011] In the context of the present application, the term "sample separation device" can in particular represent any apparatus capable of separating different fractions of a fluid sample by applying a certain separation technique, in particular gel electrophoresis or liquid chromatography.

[0012] In the context of the present application, the term "immobilizing a fluid sample" can in particular represent restricting or constraining the freedom of a fluid sample to move freely in space in any desired direction. Thus, an immobilized fluid sample can not be free to move spontaneously with respect to the immobilizing immobilizer. Immobilizing a fluid sample can thus be reducing or inhibiting the migratory properties of a fluid sample, up to completely prohibiting any movement of the fluid sample that could otherwise lead to an expansion of the fluid sample. For example, immobilizing a fluid sample can be accomplished by converting the fluid sample and / or the medium carrying or surrounding the fluid sample into a phase in which the particles cannot move freely (e.g. by gelling or freezing the sample and / or its surrounding into a gel phase or a solid phase).

[0013] In the context of the present application, the term "immobilizer" can in particular represent a medium (in particular a substance or a plurality of functionally cooperating substances) capable of triggering the immobilization of a fluid sample. Such immobilizer can be a single component that changes its own and / or the phase state of the fluid sample upon contact or interaction with the fluid sample. However, the immobilizer can also comprise a first component (preferably to be mixed with the fluid sample) and a second component. The second component can be configured such that it triggers the immobilization when it comes into contact or interaction with the first component. The fluid sample can be immobilized when the components are immobilized when the fluid sample is spatially close to the components upon interaction.

[0014] In the context of the present application, the term "immobilization unit" can in particular represent a member, a kit or a mechanism capable of triggering the immobilization of a fluid sample.

[0015] According to one exemplary embodiment of the present application, a system is provided in which a fluid sample can be temporarily prevented from freely moving into the surrounding medium. This temporary immobilization of the fluid sample can be accomplished by a fixative which is spatially located so close to the sample that the immobilization by the fixative also affects the fluid sample. In the immobilized state, the fixative and the fluid sample can be safely and reliably prevented from spatially expanding or diffusing. Unrestricted and significant spatial expansion of the fluid sample can be undesirable in terms of sample separation, as it can reduce separation precision. Advantageously, some exemplary embodiments can efficiently suppress this undesirable effect. For example, prior to starting the sample separation process, a preparation process can be desirable. During the corresponding waiting phase, the fluid sample can be temporarily kept immobilized by the fixative, so that undesirable spatial expansion or diffusion of the fluid sample can be suppressed. After completion of the preparation process and start of the separation process, the sample can be removed or released from the fixative. In terms of this sample removal procedure, the fixative can remain immobilized (while the sample can be actively moved through the fixative) or the fixative can disintegrate (so that the motion-inhibiting barrier function of the fixative is lost). The latter can be a time-dependent passive disintegration process (such as degradation) or an active disintegration process by exerting a force (such as an electrodynamic force, e.g. resulting in a migration of at least one fixative component) which invalidates the motion-inhibiting barrier function. Thus, the fluid sample can be made to move freely again in a controlled manner and be separated according to the separation mechanism of the corresponding sample separation device.

[0016] In the following, further embodiments of microfluidic devices and methods will be explained.

[0017] In one embodiment, the barrier in the form of the immobilized fixative can be actively and / or passively overcome to re-mobilize the fluid sample. For example, the barrier can be actively overcome by triggering a migration of the fluid sample through the immobilized phase or fixative. The passive overcoming of the barrier can be accomplished by, for example, dissolving the immobilized fixative or phase (due to degradation and / or diffusion). The latter dissolution can be obtained, for example, by providing thermal energy (e.g. for melting) or by providing electrodynamic energy (e.g. causing ion migration and thus the release of ionic interactions).

[0018] In one embodiment, the method comprises triggering the release by actively applying a release force, in particular by applying an electrical release force and / or a centrifugal force. Accordingly, the device can comprise a release unit for subsequently, i.e. after immobilization, at least partially releasing the fluid sample from the immobilization agent. Such a release unit can be configured for generating and applying the above-mentioned release force to the immobilized immobilization agent having the fluid sample fixed therein. For example, the release unit can be configured for releasing the fluid sample from the immobilization agent by applying an electric field. Very advantageously, an electric field generating unit already present in the electrophoretic separation device, such as a voltage source, can also be used for releasing the fluid sample. It has proven that applying an electric field to a bead or other structure comprising an immobilized immobilization agent and a (in particular electrically charged) fluid sample fixed therein surprisingly forces the particles of the fluid sample to selectively move out of the matrix of the immobilization agent and into the surrounding (in particular fluid) medium through the matrix of the immobilization agent. Thus, the fluid sample can be re-mobilized after temporary immobilization in a simple manner by applying an electric field only, in particular without additional hardware support. It has thus proven possible to force the fluid sample out of the immobilized immobilization agent by applying an electric field. This is a simple, efficient and highly selective way of removing the fluid sample from the immobilization agent. Alternatively, the release force can be different from an electrical force, for example it can be a magnetic force.

[0019] In one embodiment, the method comprises actively triggering the release by at least partially disintegrating the immobilization agent (in particular by thermal, electrical and / or chemical means). Correspondingly, the device can comprise a disintegration unit which can be configured to trigger at least partial disintegration of the immobilization agent after immobilization, thereby releasing the fluid sample. In the context of the present application, the term "disintegration" of the immobilization agent can in particular represent a process of intentionally removing, destroying, dissolving, transforming or otherwise manipulating at least a portion of the immobilization agent under immobilization conditions such that the immobilization agent no longer acts as a barrier to the movement of the fluid sample. For example, the immobilization agent can in one of its states form a matrix and / or shell of the fluid sample, thereby preventing spatial expansion or divergence of the fluid sample. Upon disintegration of the immobilization agent from this state, this inhibition of spatial expansion or divergence of the fluid sample can be partially or completely terminated. This can be achieved by, for example, a phase transition of the immobilization agent from a solid to another phase, enabling the fluid sample to move freely or substantially freely. For example, the immobilization agent can be solidified by cooling for immobilization, e.g. using a Peltier element. To release or remove the fluid sample from the immobilization agent, the immobilization agent can be heated to become liquid, thereby disintegrating the previously solid immobilization agent. Thus, the disintegration can be thermally triggered, e.g. by raising the temperature of the immobilized immobilization agent above the melting point, thereby causing it to become liquid. Also, one or more chemical agents can be added which are configured for dissolving or liquefying the immobilized immobilization agent in order to disintegrate. Also, ion interactions that reversibly immobilize can be weakened or eliminated by electrostatic forces. This can be due to the fact that ions can have a tendency to migrate in an electric field towards the respective pole (in particular depending on the strength of the electric field).

[0020] In one embodiment, the method comprises at least partially releasing the fluid sample from the immobilization agent by passively waiting for a spontaneous disintegration of the immobilization agent. It has been shown that immobilized immobilization agents disintegrate on their own within a certain time constant (e.g. typically within a few tens of minutes).

[0021] In one embodiment, immobilization comprises embedding at least part of the fluid sample into the immobilization agent and / or encapsulating at least part of the fluid sample by the immobilization agent. In other words, the immobilization agent can form a continuous spatial matrix into which the fluid sample can be accommodated. When the immobilization agent is subsequently transformed into an immovable phase, the movement of the embedded fluid sample is also prevented. However, additionally or alternatively, the immobilization agent can form a shell around the fluid sample, wherein the shell can be impermeable to the fluid sample (without a release force) (e.g. when the shell is a solid and generally impermeable). Furthermore, such a shell that can be positioned sealingly close around the fluid sample can reliably prevent spatial expansion or other diffusion or dispersion effects of the fluid sample. Whether the immobilization agent forms a continuous matrix or a hollow shell can depend on the chemical composition of the immobilization agent components, the surrounding medium, the properties of the sample, process control, etc.

[0022] In one embodiment, the method comprises triggering an immobilization phase transition of the immobilization agent to immobilize the fluid sample. In the present context, the term "immobilization phase transition" can in particular represent a phase transition of the immobilization agent (in particular in combination with the fluid sample) that activates the immobilization state of the fluid sample. For example, for the above-mentioned purposes, the immobilization agent can be transformed from a previous liquid state into a gel state or a solid state. The phase transition of the immobilization agent can then result in the immobilization of the fluid sample.

[0023] In one embodiment, the method comprises triggering the immobilization phase transition of the immobilization agent to be one of the group consisting of a solid phase, a gel phase and a highly viscous phase. In a solid, a gel or other phase with a very high viscosity having a very small pore diameter, the fluid sample cannot move freely, which equals a temporary immobilization of the fluid sample. The immobilization phase transition can be triggered externally, for example by triggering a chemical reaction, by changing the temperature (in particular by lowering the temperature) and / or the pressure, by changing the pH value, by applying a magnetic field, etc.

[0024] In one embodiment, the method comprises triggering a disintegration phase transition of the immobilization agent to disintegrate the immobilization agent. In the context of the present invention, the term "disintegration phase transition" can in particular represent a phase transition of the immobilization agent by which its function as a barrier to the fluid sample as a matrix or shell disintegrates, is destroyed or removed. In other words, the disintegration phase transition can transform the immobilization agent and thus the fluid sample interacting therewith from an immobilized state into a movable or flowable state.

[0025] In one embodiment, the mentioned disintegration can be facilitated or triggered by the addition of one or more release agents. One example of such an embodiment is the use of a substance that is capable of gelling at a suitable pH, wherein the substance can be converted from the gel phase back to another phase (e.g. a liquid phase) by providing a suitable release agent (such as an acid or a base). In another embodiment, an additive can also be provided that is capable of gelling.

[0026] In one embodiment, the method comprises triggering a disintegration phase transition of the immobilization agent into one of the group consisting of a liquid phase, a gas phase and a low viscosity phase. The disintegration phase transition can thus convert the immobilization agent into a liquid, a gas or another phase with low viscosity, such that the immobilization agent itself as well as the fluid sample therein are again free to move.

[0027] In one embodiment, the disintegration phase transition for disintegrating the immobilization agent is opposite to the immobilization phase transition of the immobilization agent for immobilizing the fluid sample. In other words, the immobilization phase transition can convert the immobilization agent and / or the fluid sample interacting therewith from a movable state or phase into an immovable state or phase. The reverse process of converting the immobilization agent and / or the fluid sample therein back into a movable or flowable state by performing a disintegration phase transition will return the immobilization agent and the fluid sample to the state before the immobilization phase transition was performed. The immobilization process can thus be reversible one or more times.

[0028] In one embodiment, at least partial immobilization of the fluid sample is performed before and / or after separation of the fluid sample. When the fluid sample is immobilized before separation, a waiting time can for example be bridged until the sample separation is started without the risk of a spatial expansion of the fluid sample due to diffusion or the like. However, the fluid sample can also be immobilized or selectively immobilized fractions or parts thereof after the separation procedure or parts thereof have been completed. When a fraction of the fluid sample has already been separated from the remaining fluid sample, only this fraction is immobilized by the immobilization agent, which keeps the respective sample part together. This is for example advantageous in terms of fractionation. It is also possible to temporarily immobilize a fluid sample that has already been separated in order to perform a feasible further analysis after the individual components or fractions have been separated. In this case, a waiting time during detection also does not lead to an unwanted spatial expansion of the separated fluid sample. It is also possible to test a re-immobilized separated sample fraction compared to a further injected fluid sample. This can be advantageous, again in terms of Western blotting.

[0029] In one embodiment, the method comprises forming at least one bead (such as a spherical body or a body or structure having another shape) consisting of the fluid sample and a fixative for immobilizing the fluid sample. In the context of the present application, the term "bead" can in particular represent a liquid droplet separated by phase transition (descriptively in a similar way as a ball on a trampoline) from the surrounding medium (such as a separation medium). However, the bead can also be a gel sphere in which the sample is present and which is separated by phase transition with respect to the surrounding medium (such as a separation medium). Such a bead can also be a liquid droplet which is separated by a gel skin around the droplet with respect to the surrounding medium (such as a separation medium) while the fluid sample is still inside the gel skin in liquid phase. Such a bead can also be a frozen droplet of the fluid sample or a frozen droplet containing the fluid sample. Further, the bead can be a solid to which the fluid sample is adhered. For example, the bead can have a maximum dimension of less than 5 mm, in particular of less than 3 mm, more in particular of less than 1 mm. In such an embodiment, the bead-like combination of the fixative and the fluid sample can be a confined physical structure by which the fluid sample is kept together as a whole in space within a limited spatial extent.

[0030] In one embodiment, the method comprises shielding the immobilized fluid sample from a (e.g. fluid, in particular liquid and / or gaseous or gel-like) medium in the environment of the fixative during the immobilization of the fluid sample. For example, in the case of a gel electrophoresis, the surrounding medium can be a gel matrix or, in the case of a liquid chromatography, the surrounding medium can be a liquid mobile phase (such as a solvent or a solvent composition). While the fluid sample can be able to move in such a medium under normal circumstances, the immobilization caused by the fixative temporarily prevents such a free movement until the fixative is subsequently disintegrated or the fluid sample is removed from the fixative in another way (e.g. by an electrical force).

[0031] In one embodiment, the method comprises contacting the fluid sample with the medium by releasing the fluid sample from the fixative. Thus, the process of disintegrating the fixative or passing the fluid sample through the immobilized fixative can trigger the physical contact of the fluid sample with the medium and, thus, can activate the ability of the fluid sample to move in the fluid or gel-like medium (e.g. for separating different fractions of the fluid sample).

[0032] In one embodiment, the fluid sample is a biological sample. Such a biological sample can be particularly sensitive and prone to spatial expansion during a waiting phase, e.g. a waiting phase required for preparing the fluid sample for analysis in a sample separation device. For example, the biological sample can comprise separable proteins and / or DNA. Examples of such a biological sample are a blood sample, orange juice, a biochemical preparation, a metabolite, a body fluid after a digestion drug, etc.

[0033] In one embodiment, the method can comprise (after immobilization) preparing the fluid sample for a subsequent separation or analysis, and (after preparation and after release) performing the separation or analysis of the fluid sample. After release, removal or disintegration, the fluid sample is again able to move freely, in particular in terms of analysis or sample separation. Such separation can for example be performed by electrophoresis (in particular gel electrophoresis) or chromatography (in particular liquid chromatography). In gel electrophoresis, it can be desirable that a fluid sample injected into a gel-type separation medium needs to wait (e.g. for one minute) until a preparation for starting the actual separation process is completed (one example for such a preparation process in terms of gel electrophoresis is the formation of an oil film). During the respective waiting time, immobilization of the fluid sample can prevent undesired spatial expansion of the fluid sample due to effects such as strong diffusion. In liquid chromatography, an introduced fluid sample can be temporarily stored in a sample loop or other sample containing volume, wherein the fluid sample can only fill a part of the sample containing volume. At an appropriate point in time, a fluid valve can be switched to inject the introduced and temporarily stored fluid sample from the sample containing volume into a flow path between a fluid driver and a sample separation unit. By temporarily immobilizing the fluid sample in the immobilization agent while the fluid sample is in the sample containing volume, undesired spatial expansion of the fluid sample during the waiting time before injection can be avoided. However, other separation techniques can also be applied. Moreover, other analysis procedures can also be performed, e.g. determining a component concentration of the fluid sample, or determining any other property of the fluid sample.

[0034] In one embodiment, the method comprises applying the fluid sample on a planar carrier, and subsequently applying a separation medium on the planar carrier and interacting with the fluid sample. Such a concept can be advantageous in terms of open microfluidic structures.

[0035] In one embodiment, the immobilization agent comprises a first component and a second component (which can initially be stored separately from the first component), the first component and the second component being configured such that the immobilization agent is immobilized by an interaction (in particular by a chemical reaction) between the first component and the second component. The method can comprise triggering the immobilization by mixing the fluid sample with the first component, and then injecting the mixture into a medium comprising the second component. When the first component is mixed with the fluid sample prior to this injection and thus prior to the immobilization, it can be ensured that the subsequent addition of the second component (which can trigger the immobilization) takes place in a state where the sample is already in close spatial relationship with the first component. It can thus be ensured that triggering the immobilization of the immobilization agent will also significantly reduce the mobility of the fluid sample located within the range of the immobilization.

[0036] In one embodiment, the medium carrying or comprising the second component comprises a gel (e.g. an electrophoretic gel, in particular a polyacrylamide-based gel) and / or a mobile phase (e.g. a chromatographic mobile phase, such as a solvent or a solvent composition, e.g. comprising an organic component, like methanol or acetonitrile, and an inorganic component, such as water). Thus, the medium can be selected in accordance with the separation technique used for separating the fluid sample into fractions.

[0037] In one embodiment, the first component is calcium chloride or calcium gluco lactate. However, salts containing another alkaline earth metal than calcium and / or another halogen than chlorine can also be used. In one embodiment, the second component is sodium alginate. However, sodium can also be replaced by another alkali metal or the like. Other combinations of the first component and the second component are also possible. The first component and the second component should be chosen such that only their interaction can trigger the immobilization process. They can further be configured such that the immobilization process is reversible, i.e. the disintegration of the immobilized immobilization agent can subsequently be achieved by an external stimulus or by an intrinsic disintegration behavior of the immobilization agent having a sufficiently long time constant.

[0038] In one embodiment, the interaction triggering the immobilization is an ion exchange between the first component and the second component. The term "ion exchange" can represent an ion exchange between two chemicals in the form of the first component and the second component. More particularly, the sodium ions of sodium alginate can be replaced by calcium ions of calcium chloride. This anion exchange can trigger the formation of a gel phase of the immobilization agent, in which the fluid sample can be immobilized.

[0039] In one embodiment, the interaction triggering the immobilization is a hydrogel formation under participation of the first component and the second component. In the context of the present application, the term "hydrogel" can particularly represent a network of hydrophilic polymer chains, e.g. a colloid gel, in which water is the dispersion medium. Hydrogels can be highly absorbent (they can contain more than 90% water) natural or synthetic polymer networks. The formation of a hydrogel in which the fluid sample is embedded is one embodiment of the immobilization process preventing the spatial expansion of the sample plug.

[0040] In another embodiment, the first component is thrombin and the second component is fibrinogen. The thrombin-catalyzed conversion of fibrinogen to fibrin can involve three reversible steps, with thrombin participating only in the first step, which is a limited proteolysis to release a fibrinopeptide from fibrinogen, resulting in a fibrin monomer. In the second step, the fibrin monomers form an intermediate polymer through non-covalent interactions. In the third step, the intermediate polymer aggregates to form a fibrin clot. In this embodiment, the fluid sample can be immobilized within such a clot. Since the process is reversible, the fluid sample can be selectively immobilized in the fibrin bead or released from the fibrin. Thus, the above-described process involving sodium alginate and calcium chloride is only one (but a preferred) embodiment and example of fluid sample immobilization according to one embodiment of the present application. For example, one of thrombin and fibrinogen can be mixed (or pre-mixed) with the fluid sample, while the other of thrombin and fibrinogen can be mixed (or pre-mixed) with the separation medium (e.g., the gel in the gel electrophoresis example or the solvent composition in the liquid chromatography example).

[0041] In another embodiment, the first component is a polymerizable substance and the second component is a cross-linking agent, or vice versa. More generally, the immobilization process can be achieved through in situ polymerization. For example, a polymerizable substance can be added to the separation medium, which polymerizes upon contact with a cross-linking agent (which can be mixed with the fluid sample) to immobilize or immobilize the fluid sample. In another embodiment, such polymerization of the polymerizable immobilization agent can be triggered by another stimulus mechanism, such as the supply of ultraviolet radiation, thermal energy, a chemical reagent, etc. The polymerization can also be triggered only locally.

[0042] It should be noted, however, that exemplary embodiments of the present application can use a variety of mechanisms to immobilize the immobilization agent and the fluid sample that interacts with such immobilization agent or portion thereof.

[0043] The following table gives an overview of immobilization agents and their components that can be used according to some different exemplary embodiments of the present application:

[0044]

[0045]

[0046] In one embodiment, the method comprises providing a separation medium on a carrier for separating the fluid sample, injecting the fluid sample into the separation medium (in particular triggering immobilization thereby), and separating the fluid sample by the separation medium (in particular by electrophoresis) after immobilization and release. For example, such a separation medium can be a gel strip or matrix in the case of gel electrophoresis, in particular applied (e.g. printed) on a carrier. In the case of liquid chromatography, the separation medium can be a chromatographic separation medium in or on a carrier such as a column tube. When the immobilization of the fluid sample is removed, the fluid sample is again freely movable and thus can be separated into different fractions by the separation medium.

[0047] In one embodiment, the method comprises locally immobilizing the fluid sample by the immobilization agent only while the environment of the fluid sample and the immobilization agent is kept in an unchanged (e.g. fluid) state. Thus, in such an embodiment, the temporary immobilization essentially only affects the fluid sample and the immobilization agent itself while the environment is kept unchanged (e.g. fluid) and thus also freely movable during the maintenance of the immobilized state. This ensures that the separation device is only affected to the minimum and absolutely necessary extent by the immobilization process (i.e. the extent involving the fluid sample). According to such an embodiment, the period of time during which the fluid sample remains immobilized can be used for preparing a subsequent analysis or separation, for completing a previous analysis or separation, etc. Thus, the fluid sample is prevented from undesirably spatially expanding or spatially diverging during the respective waiting time, thereby avoiding an undesired dilution or spatial expansion of the fluid sample.

[0048] However, in other embodiments, the entire system can be temporarily immobilized (rather than only a small portion of it being locally immobilized).

[0049] In one embodiment, the device comprises an electric field generating unit configured for generating an electric field for applying an electric force to the fluid sample in the medium during and / or after the release, in particular for releasing the fluid sample from the immobilized immobilization agent and / or for separating and moving the fluid sample towards a detector for detecting separated fractions of the fluid sample. The electric field generating unit can be used to trigger the release of the fluid sample from the immobilized immobilization agent by an electric release force. Furthermore, the separation process in terms of gel electrophoresis for separating the released fluid sample can differentially move different fractions of the fluid sample in the electric field generated by the (preferably the same) electric field generating unit, thereby enabling the separation of fractions of the fluid sample.

[0050] In one embodiment, the device comprises an injection unit for injecting the at least partially immobilized fluid sample into a separation channel (e.g. the separation channel in which the fluid sample is to be separated or a separation channel for directing the fluid sample into a sample separation unit for separation). Prior to, during and / or after such an injection process, the previously immobilized fluid sample can be re-mobilized and thus prepared for a subsequent separation. It is also possible to form a series of immobilized fluid sample beads in a supply channel and inject the fluid sample beads one after the other into a separation channel for separation into fractions.

[0051] In one embodiment, the device comprises a compensation unit configured for (in particular computationally) at least partially compensating for deviations between separation results in the presence of a fixation agent and separation results in the absence of a fixation agent. Very advantageously, fixation agents are disclosed which have only very little influence on the chemical environment of the fluid sample during the pre-separation immobilization phase (e.g. a combination of sodium alginate and calcium chloride). However, in the case of fixation agents which have a significant influence on the separation results are employed in some exemplary embodiments, the deviations (artifacts) of the separation results due to the chemical influence of the fixation agent can be partially or completely eliminated by compensating or correcting the separation results according to the above influences. As a basis for such a compensation or correction, the behavior of the fluid sample in the presence of a chemical environment of a fixation agent can be modeled compared to the behavior in the absence of a fixation agent. In addition or alternatively, such a compensation can be done by using reference measurements (e.g. with and without fixation agent) which can be stored in e.g. a database or lookup table. Furthermore, expert knowledge (e.g. expert rules) can also be employed for this purpose. By taking this measure, the accuracy of the separation results can be further improved.

[0052] In one embodiment, the device comprises a fluid driver configured for driving the fluid sample in the mobile phase after release towards the sample separation unit, and the device comprises a sample separation unit which can be configured for separating the fluid sample. For example, such a fluid driver can be a high-pressure pump configured for driving the mobile phase (i.e. a solvent or a solvent composition) and the fluid sample towards the sample separation unit. The sample separation unit can be a chromatographic column. Thus, the arrangement corresponds to a sample analysis by liquid chromatography.

[0053] In one embodiment, the device is a microfluidic device. In the context of the present application, the term "microfluidic device" can in particular represent a device capable of analyzing or processing a fluid sample having channels of the order of micrometers in diameter. The flow rate can also be in the range of microliters per minute. Such a microfluidic device can for example be a liquid chromatography device or a gel electrophoresis device capable of processing small amounts of fluid sample.

[0054] Some embodiments of the present application can be implemented in a routinely available HPLC system, such as the Agilent 1200 Series Rapid Resolution LC System or the Agilent 120 HPLC Series (both provided by the Applicant, Agilent Technologies, Inc.).

[0055] One embodiment of a sample separation device includes a pumping apparatus as a fluid driver or mobile phase driver, the pumping apparatus having a pump piston for reciprocating motion in a pump working chamber to compress a liquid in the pump working chamber to a high pressure at which the compressibility of the liquid becomes significant. The pumping apparatus can be configured to know (through input by an operator, a notification from another module of the instrument, or the like) or otherwise obtain solvent properties that can be used to represent or retrieve actual characteristics of the fluid contents (as expected in the sampling apparatus).

[0056] The separation unit of the fluid separation apparatus preferably includes a chromatographic column providing a stationary phase. The column can be a glass or steel tube (e.g., 50 pm to 5 mm in diameter, 1 cm to 1 m in length) or a microfluidic column (disclosed in, e.g., EP 1577012 or the Agilent 1200 Series HPLC-on-Chip / MS system provided by the Applicant, Agilent Technologies, Inc.). The individual components are held differentially by the stationary phase and at least partially separated from each other while they are transported through the column with the eluent at different speeds. At the end of the column, they are eluted one at a time or at least not completely simultaneously. The eluent can also be collected into a series of fractions throughout the chromatography process. The stationary phase or adsorbent in column chromatography is usually a solid material. The most common stationary phase for column chromatography is silica gel, surface-modified silica gel, then alumina. Cellulose powder was often used in the past. It can also be ion exchange chromatography, reverse phase chromatography (RP), affinity chromatography, or expanded bed adsorption (EBA). The stationary phase is usually a finely ground powder or gel and / or microporous to increase the surface.

[0057] The mobile phase (or eluent) can be a pure solvent or a mixture of different solvents, such as water and an organic solvent, such as ACN, acetonitrile. For example, a selection can be made to minimize the retention of the target compound and / or the amount of mobile phase to run the chromatography. The mobile phase can also be selected so that different compounds or fractions of the fluid sample can be effectively separated. The mobile phase can comprise an organic solvent, such as methanol or acetonitrile, typically diluted with water. For gradient operation, water and organic are delivered in separate bottles, and a gradient pump delivers a programmed blend from these bottles to the system. Other commonly used solvents can be isopropanol, THF, hexane, ethanol, and / or any combination thereof, or any combination of these solvents with the above-mentioned solvents.

[0058] The fluid sample can include, but is not limited to, any type of process liquid, natural sample (if juice), body fluid (such as blood plasma), or it can be a result of a reaction, such as from a fermentation broth.

[0059] The pressure generated by the fluid driver of the mobile phase can be in the range of 2-200 MPa (20 bar to 2000 bar), in particular in the range of 10-150 MPa (20 bar to 1500 bar), more in particular in the range of 50-120 MPa (500 bar to 1200 bar).

[0060] The sample separation device, such as an HPLC system, can further comprise a detector for detecting the separated compounds of the fluid sample, a fractionation unit for outputting the separated compounds of the fluid sample, or any combination thereof. Further details of such an HPLC system are disclosed with respect to the Agilent 1200 Series Rapid Resolution LC System or the Agilent 1200 HPLC Series, both provided by the applicant Agilent Technologies, Inc.

[0061] Embodiments of the present application can be partially or entirely implemented by one or more suitable software programs, which can be stored or otherwise provided on any kind of data carrier, and which can be executed in or by any suitable data processing unit. The software programs or routines can preferably be applied in or by the control unit. BRIEF DESCRIPTION OF DRAWINGS

[0062] Other objects and many of the attendant advantages of embodiments of the present application will be readily appreciated as the same becomes better understood by reference to the following more detailed description of the embodiments when considered in connection with the accompanying drawings, wherein: like reference numerals designate identical components throughout the figures. Before embodiments of the present application are disclosed and described, it is to be understood that the application is not limited to the specific

[0063] Figure 1A liquid sample separation device according to some embodiments of the application is shown, in particular for high performance liquid chromatography (HPLC).

[0064] Figure 2 A liquid sample separation device according to some embodiments of the application is shown, in particular for gel electrophoresis.

[0065] Figure 2A A three-dimensional view of a part of a liquid sample separation device according to some embodiments of the application is shown, demonstrating the injection of a fluid sample into the separation channel. Figure 2 Similar embodiments and some alternatives are shown.

[0066] Figure 3 A sample separation device according to one exemplary embodiment of the application is shown schematically.

[0067] Figure 4 A part of a sample separation device according to one exemplary embodiment of the application is shown, wherein a number of gel beads with immobilized fluid sample are injected sequentially into the separation channel for subsequent separation of the fluid sample in each one of the beads.

[0068] Figure 5 A fluid sample injected into the channel without spatial focusing is shown.

[0069] Figure 6 A separation medium with spatially focused fluid sample according to one exemplary embodiment of the application is shown.

[0070] Figure 7 A spatially focused fluid sample during spatial focusing and at a later point in time according to one exemplary embodiment of the application is shown.

[0071] Figure 8 Separation of a spatially focused fluid sample according to one exemplary embodiment of the application is shown.

[0072] The illustrations in the drawings are schematic. DETAILED DESCRIPTION

[0073] Before the drawings are further described in detail, some basic considerations of the present application will be summarized based on some exemplary embodiments that have been developed.

[0074] According to one exemplary embodiment of the application, a delayed sample injection can be accomplished by a locally and temporarily created immobilization of the fluid sample (in particular by a phase transition trigger). More particularly, a method for sample injection or sample storage by locally created phase transition is provided.

[0075] According to one exemplary embodiment, a method is provided for temporarily storing a biological or other fluid sample in a (micro)fluidic analysis system, more particularly a sample separation device. The fluid sample can first be separated from the remaining fluidic system by a controlled in situ phase transition. In a subsequent procedure, the sample can be restored to interact with the fluidic analysis system, e.g. by reversing the phase transition or by passing through the substance that has undergone the phase transition.

[0076] In this context, the biological sample can particularly be a water-based liquid containing biological matter such as proteins, lipids, DNA, RNA, etc. Such a fluid sample can be subjected to a separation process to analyze its composition, can be purified, can be reacted with one or more other substances, etc.

[0077] The microfluidic (analysis) system can be a fluidic analysis system composed of fluidic components such as channels, mixers, etc. and can be used for the purpose of preparing fluid, particularly biological, samples and / or analyzing fluid, particularly biological, samples. Examples of such analysis are electrophoresis, chromatography, etc. These devices can comprise a closed microfluidic system or an open microfluidic system, or even a mixture of both concepts.

[0078] Injection can represent the intentional insertion of a fluid, particularly biological, sample in a fluidic analysis system, more particularly a sample separation device.

[0079] Sample storage can represent the process of storing biological samples in a fluidic analysis system such as a sample separation device so that they can be actively injected and analyzed in response to a control command. Such a control command can trigger the disintegration or removal of a fluid sample from a previously immobilized immobilization agent that interacts with the fluid sample.

[0080] Immobilization of a fluid sample by a corresponding phase transition and removal of the fluid sample from the immobilization agent by disintegration of the immobilization agent can be triggered by the phase transition. Such a phase transition can be a transition of a material between different aggregation states, particularly a solid or solid phase, a liquid or liquid phase and a gaseous or gas phase. More generally, a phase transition can also cover a transition that changes a specific material property. For example, a transition into a superconducting state or the formation of a hydrogel by polymerization can also represent a phase transition.

[0081] The embodiments of the present invention described below describe an example of a gel transition of a hydrogel, more particularly a reaction between sodium alginate and calcium chloride. The gel formation of sodium alginate produced by algae can be triggered by replacing sodium by calcium ions, i.e. by ion exchange. Descriptively and without wishing to be bound by a specific theory, it is currently assumed that the alginate thereby forms a three-dimensional polymer network with interstices that are filled with a liquid, particularly water, and can be denoted as a hydrogel.

[0082] Some exemplary embodiments of the present application use the mentioned phase transition and optionally a further counter effect in order to store a fluid sample in a microfluidic system in a focused condition for subsequent insertion or injection of the sample in an active or passive process. For this purpose, a fluid sample, in particular a biological sample, can be mixed with calcium chloride as a first component of a fixation agent and inserted into a sample separation device, which can be a microfluidic analysis system, containing sodium alginate as a second component of the fixation agent. Once contact between the first and second component of the fixation agent is established, a hydrogel is formed due to the interaction between sodium alginate and calcium chloride. This hydrogel surrounds and / or embeds the fluid sample, thereby separating the fluid sample from the fluid environment. Descriptively, the formed hydrogel barrier significantly and instantaneously slows down the interaction between the fluid sample and the fluid system, which otherwise could take place immediately in the presence of two completely liquid phases in direct contact with each other. By taking this measure, the fluid sample can be temporarily stored in a focused and fixed state, in which the ordinary ability and tendency of the fluid sample to diffuse and thereby spatially expand in the fluid environment is significantly reduced. For example, in case a time-consuming preparation procedure (e.g. supplying further samples, reagents, etc.) is required before the fluid sample is analyzed or separated, the storage of such a temporarily immobilized fluid sample is highly advantageous, as it prevents smearing of the fluid sample during the waiting time. Additionally, a local binding and / or fixation of the sample to the separation device can be established to increase the robustness against pressure driven flow phenomena that can occur and are often observed in such microfluidic networks.

[0083] Furthermore, in conventional methods the accuracy and strength of the detection signal can deteriorate due to the time delay between providing the sample and performing the separation or analysis. The reason for this time delay is for example the delay in applying the driving force (e.g. electrostatic force, centrifugal force, pressure, temperature, etc.) for the analysis and the time required for preparing the analysis (e.g. adjusting and aligning the focus of the detector or optical elements).

[0084] According to one exemplary embodiment of the present application, the immobilization of the fluid sample allows to pass the mentioned time delay without deteriorating the detection signal. This is a consequence of the strong inhibition of the spatial expansion of the fluid sample during storage due to the temporary immobilization. In addition to the passive injection by diffusion of the hydrogel, the driving force for the analysis / separation and / or other release forces can actively allow for the injection of the fluid sample (e.g. by applying an electrostatic force and thus the migration of the charged biomolecules through the hydrogel barrier, or the ionic dissolution or disintegration of the hydrogel barrier).

[0085] One exemplary embodiment of the present invention relates to electrophoresis in an open microfluidic system. Electrophoresis of charged biomolecules can be performed in a microchannel filled with a separation medium. After injection of the biomolecules in the microchannel, fractions of the fluid sample are separated due to an electric field applied along the channel, more particularly according to different sizes of the fractions. Electrophoresis is particularly advantageous when performed in an open microfluidic system. In such embodiments, the separation medium applied on the planar carrier replaces the separation medium applied to the microchannel.

[0086] The fluid sample to be separated or analyzed can be injected in a non-contacting manner. Because a period of time can elapse between injection of the fluid sample and analysis or separation of the fluid sample (e.g. due to alignment of detection optics, application of a separation voltage, etc.), the fluid sample can experience strong and unconfined diffusion due to flow along the tubing or due to impact of the free flying droplet of the non-contact dispensing of the fluid sample on the separation medium, particularly in case the droplet diameter exceeds the height of the tubing. This spatial expansion can also broaden the detected peaks after separation of the fluid sample. This reduces the resolution of the separation or more generally the resolution of the analysis of the fluid sample.

[0087] By applying calcium chloride to the fluid sample and by applying sodium alginate to the separation medium, descriptively each sample droplet rich in calcium chloride (or another first component of the fixative) can be frozen in situ (more precisely can be embedded in and / or surrounded by a gel matrix) upon reaching the separation medium rich in sodium alginate (or another second component of the fixative). Due to the calcium chloride in the fluid sample, a portion of the channel forms a hydrogel and separates the sample droplet from the separation medium.

[0088] As a result, the fluid sample bead can thus be spatially focused in the channel. The fluid sample treated with the fixative in the described manner is thus not spatially expanded or diverged and has a size that is fully or substantially identical to the bead diameter established, for example, during dispensing. Experiments have shown that the spatial extent of the fluid sample presence only increases very little or even minimally, even after a significant waiting time of, for example, 20 minutes. The small increase in the spatial extent of the fluid sample presence can be due to influences such as diffusion through the hydrogel and some dissolution of the hydrogel over time.

[0089] Reference will now be made in detail to the drawings identified above, Figure 1A general schematic of a liquid separation system depicting an example of a sample separation apparatus 10 according to an example embodiment of the application. A pump as a fluid driver 20 receives a mobile phase from a solvent supply 25, typically via a degassing device 27 that degasses and thus reduces the amount of dissolved gas in the mobile phase. The mobile phase drive or fluid driver 20 drives the mobile phase through a sample separation unit 30, such as a chromatographic column, containing a stationary phase. A sampler or injection unit 40 employing a fluid valve 95 can be provided between the fluid driver 20 and the separation unit 30 to introduce or add, often referred to as sample introduction, a sample fluid into the mobile phase. The stationary phase of the separation unit 30 is configured to separate compounds of the sample liquid. A detector 50 is provided to detect the separated compounds of the sample fluid. A fractioner unit 60 can be provided to output the separated compounds of the sample fluid.

[0090] While the mobile phase can consist of only one solvent, it can also be a mixture of multiple solvents. This mixing can be low pressure mixing and provided upstream of the fluid driver 20 so that the fluid driver 20 receives and pumps the mixed solvents as the mobile phase. Alternatively, the fluid driver 20 can consist of multiple separate pumping units, each receiving and pumping a different solvent or mixture. The composition of the mobile phase (mixture) can remain constant over time, so-called isocratic mode, or vary over time, so-called gradient mode.

[0091] The data processing unit or control unit 70 can be a PC or workstation which can be coupled (as indicated by the dashed arrows) to one or more components in the sample separation apparatus 10 in order to receive information and / or control operation. For example, the control unit 70 can control operation of the fluid drive 20 (e.g. set control parameters) and receive information therefrom regarding actual working conditions such as output pressure at the outlet of the pump etc. The control unit 70 can also control operation of the solvent supply 25 (e.g. set the solvent or solvent mixture to be supplied) and / or the degassing device 27 (e.g. set control parameters such as vacuum level) and can receive information therefrom regarding actual working conditions such as the composition of the solvent supplied over time, the vacuum level etc. The control unit 70 can further control operation of the sampling unit or injection unit 40 (e.g. control sample injection or synchronize sample injection with operating conditions of the fluid drive 20). The separation unit 30 can also be controlled by the control unit 70 (e.g. select a particular flow path or column, set operating temperature etc.) and send information (e.g. operating conditions) to the control unit 70 (as return). The detector 50 can be controlled by the control unit 70 (e.g. regarding spectral or wavelength settings, set time constants, start / stop data acquisition) and send information (e.g. regarding detected sample compounds) to the control unit 70 as well. The control unit 70 can also control operation of the fractionation unit 60 (e.g. in combination with data received from the detector 50) and provide data as return.

[0092] Figure 1 It is also schematically shown how the sample containment volume 94 (such as a sample loop) can be filled with the fluid sample 100. For example, the needle 91 can be temporarily driven out of its seat (not shown) in the injection unit 40 and can be temporarily immersed (see reference numeral 96) into the fluid sample 100 in the vial or other fluid container 93. An aliquot of the fluid sample 100 can then be sucked via the needle 91 into the sample containment volume 94. Figure 1

[0093] The described procedure can involve that the fluid sample 100 which needs to fill only a portion of the sample containment volume 94, for example, waits for a certain waiting time between completion of the suction process from the fluid container 93 into the sample containment volume 94 and injection of the fluid sample 100 from the sample containment volume 94 into the flow path between the fluid drive 20 and the separation unit 30. During this waiting time, the fluid sample 100 can spatially expand within the spatial extension of the sample containment volume 94. This can lead to a reduction of the accuracy of the separation process.

[0094] To avoid the mentioned reduction of separation accuracy, one exemplary embodiment of the present invention provides for a immobilization unit 116 in the sample separation apparatus 10. The immobilization unit 116 can be configured to immobilize the fluid sample 100 in the sample containment volume 94 during the waiting time between completion of the suction process from the fluid container 93 into the sample containment volume 94 and injection of the fluid sample 100 from the sample containment volume 94 into the flow path between the fluid drive 20 and the separation unit 30. Figure 1 ​In embodiments, the immobilization unit 116 can be configured for immobilizing the fluid sample 100 while waiting in the sample containment volume 94 for injection and subsequent separation. More particularly, the immobilization unit 116 can be configured for embedding or enclosing the fluid sample 100 in the sample containment volume 94 by the immobilization agent 102 for inhibiting spatial expansion of the fluid sample 100. In the illustrated embodiment, immobilization can be accomplished by enclosing the fluid sample 100 in the sample containment volume 94 by an enclosing shell of the immobilization agent 102, see detail 125. As shown in detail 125, one or more beads 104 can be formed that include a core with the fluid sample 100 surrounded by a shell of immobilized (e.g. gelled) immobilization agent 102. The formation of these beads 104 can take place in the sample containment volume 94, i.e. at the beginning of the waiting time. Thus, the fluid sample 100 is safely protected from spatial expansion in the sample containment volume 94 due to diffusion effects or the like during the waiting time. Descriptively, by immobilizing the fluid sample 100 in a spatially limited manner, the immobilized fluid sample 100 can be shielded against a fluidic medium (e.g. a mobile phase, i.e. a solvent or a solvent composition) in the environment of the immobilization agent 102. For activation of the immobilization, an immobilization phase transition of the immobilization agent 102 can be forced to immobilize the fluid sample 100. This can be accomplished by triggering the immobilization phase transition of the immobilization agent 102 into a gel phase. Descriptively, the fluid sample 100 will then be surrounded by a gel shell (and / or embedded in a gel matrix), preventing or inhibiting free diffusion of the fluid sample 100.

[0095] After injection of the one or more beads 104 by switching the fluidic valve 95 into the flow path between the fluid driver 20 and the separation unit 30, sample separation should be started. The mentioned flow path corresponds to the separation channel 169 in which the fluid sample 100 is separated. For being able to separate the fluid sample 100, the fluid sample 100 of the one or more beads 104 should be re-mobilized. This can be achieved by a release unit 117 and / or by a disintegration unit 118 that is arranged in the flow path between the fluid driver 20 and the separation unit 30 and that is configured for releasing the fluid sample 100 from the immobilization agent 102. Additionally or alternatively, the re-mobilization of the fluid sample 100 of the formed partial beads 104 can also be performed before transferring the fluid sample 100 from the sample containment volume 94 into the flow path, e.g. still in the sample containment volume 94 after expiration of the waiting time.

[0096] For example, the disintegration unit 118 can be configured for releasing the fluid sample 100 from the immobilized immobilizer 102 previously in the gel phase by a temperature increase that destroys the shell. By a suitable temperature increase of one or more beads 104 induced by the disintegration unit 118, the gel shell of the immobilized immobilizer 102 can turn into a liquid and thereby the fluid sample 100 previously enclosed therein can be released. Additionally or alternatively, the disintegration of the immobilized immobilizer 102 can also be triggered chemically, i.e. by adding one or more chemicals that cause the disintegration of the immobilized immobilizer 102.

[0097] Additionally or alternatively, the release of the fluid sample 100 from the immobilized immobilizer 102 can be achieved by a release unit 117. For example, the release unit 117 can trigger the release by applying an electrical release force that triggers a diffusive-like migration of at least a portion of the charged particles of the fluid sample 100 through the gel shell of the immobilized immobilizer 102. It has been shown that the application of an electrical force to the beads 104 can move the charged particles of the fluid sample 100 through the immobilized immobilizer 102 in order to release and detach from the immobilizer 102 independently for a subsequent separation.

[0098] In summary, the fluid sample 100 can be processed in the sample separation device 10 in such a way that the fluid sample is first immobilized by phase transition of the immobilizer 102 into the gel phase in order to suppress the spatial expansion of the fluid sample 100 during a waiting time until an injection is performed and the actual separation process is started. Subsequently, the fluid sample 100 is released from the immobilizer 102, for example by a further phase transition of the immobilizer 102 into the liquid phase by the disintegration unit 118 and / or by promoting a diffusion of the fluid sample 100 through the immobilizer 102 by an electrical release force applied under control of the release unit 117. After this release, the illustrated embodiment completes a chromatographic separation of the released fluid sample 100 by a chromatographic separation medium 112, such as a stationary phase, in the separation unit 30, such as a chromatographic separation column. Prior to and during the separation, the fluid sample 100 is carried in a fluid medium 106, such as a mobile phase. After the release of the fluid sample 100 from the immobilizer 102, the fluid sample 100 is in direct contact with the fluid medium 106, see detail 127.

[0099] Also as Figure 1As schematically shown, the sample separation device 10 can also comprise a compensation unit 121 (here embodied as part of the control unit 70) configured for computationally compensating for deviations between separation results in the presence of the immobilization agent 102 and separation results in the absence of the immobilization agent 102. More particularly, the presence of the immobilization agent 102 can slightly influence the chemical conditions under which the fluid sample 100 is present. This in turn can have a slight influence on the separation properties of the fluid sample 100. To avoid the influence of this and other effects on the separation accuracy of the fluid sample 100, such effects can be taken into account and compensated for by a correction calculation performed by the compensation unit 121. Reference measurements, theoretical models and / or other experimental and / or historical data as well as expert knowledge can be taken into account in the framework of such a compensation procedure.

[0100] Figure 2 A liquid sample separation device 10, in particular for gel electrophoresis, according to some embodiments of the present application is shown.

[0101] Before describing some embodiments of the present application in more detail Figure 2 Before describing some embodiments of the present application in more detail Figure 2 In some preferred embodiments, the immobilization agent 102 comprises a first component 108 (e.g. calcium chloride) and a second component 110 (e.g. sodium alginate) configured such that the immobilization agent 102 is immobilized by contact or interaction between the first component 108 and the second component 110 as this leads to an immobilization chemical reaction between the components 108, 110. Such a chemical reaction can involve an ion exchange between calcium ions of the first component 108 and sodium ions of the second component 110 and the resulting formation of a hydrogel. To ensure that the fluid sample 100 is simultaneously immobilized when the immobilization agent 102 is immobilized, it can be very advantageous to mix the fluid sample 100 with the first component 108 before adding the second component 110. This ensures that the fluid sample 100 is in close spatial relationship with the first component 108 during the immobilization process. The prepared mixture of the fluid sample 100 and the first component 108 can be applied to the medium 112 (for gel electrophoretic separation of the fluid sample 100 in embodiments of the present application) which already contains or is now provided with the second component 110. During this application, the immobilization can be triggered as soon as the first component 108 (with the fluid sample 100 mixed therewith) comes into contact with the second component 110 in the medium 112. As a result, the immobilization reaction is spatially strictly controllable. Thus, the fluid sample 100 is only locally immobilized by the immobilization agent 102, while the environment of the fluid sample 100 and the immobilization agent 102 remains in an unchanged state.

[0102] After immobilization, preparation tasks for the subsequent separation of the fluid sample 100 can be carried out (e.g. an oil film can be formed) without risking a spatially significant expansion of the fluid sample 100 during the respective waiting time. When the preparation is complete, the release of the fluid sample 100 from the matrix of the immobilizing fixative 102 can be triggered. This can be done, for example, by switching on an electric field which causes the charged particles of the fluid sample 100 to diffuse through the gel-type fixative 102 due to the applied electrical forces resulting from the electric field. After immobilization, preparation and release, the method can continue by separating the re-motile fluid sample 100 through the separation medium 112 according to the principle of gel electrophoresis.

[0103] Thus, Figure 2 An example of a gel electrophoresis analysis according to one exemplary embodiment of the present application is shown. A gel is applied as the electrophoretic separation medium 112 on a carrier 115. From Figure 2 It can be seen that the fluid sample 100 (present in a vial, pipette or contact (non-contact) dispenser of the immobilization unit 116) can have been pre-mixed with the first component 108 of the fixative 102. As can also be seen from Figure 2 It can be seen that the fluid sample 100 (present in a vial, pipette or contact (non-contact) dispenser of the immobilization unit 116) can have been pre-mixed with the first component 108 of the fixative 102. As can also be seen from Figure 2 It can be seen that the fluid sample 100 (present in a vial, pipette or contact (non-contact) dispenser of the immobilization unit 116) can have been pre-mixed with the first component 108 of the fixative 102. As can also be seen from

[0104] In a passive implementation, one can simply wait until the hydrogel has disintegrated or the fluid sample 100 has diffused through the hydrogel material into the surrounding fluid medium, i.e. the separation medium 112. In an active implementation, an electric field can be applied to force the charged particles of the fluid sample 100 to move through the hydrogel under the influence of an electric release force (e.g. generated by applying a voltage by the voltage source 180 operating the electrophoresis electrodes 182, 184). The electric field generated by the electric field generation unit 123 (constituted by the voltage source 180 and the electrodes 182, 184) thus generates a release force acting on the fluid sample 100 in the beads 104. Advantageously, the electric field generation unit 123 is configured for generating an electric field for applying an electric force to the fluid sample 100 in the medium 112 during and after the release. The mentioned electric field causes the release or removal of the charged particles of the fluid sample 100 from the immobilized immobilizer 102. Advantageously, the mentioned electric field also causes the released charged particles of the fluid sample 100 to move towards the detector 50 for detecting the separated fractions of the fluid sample 100. In other words, the electric field generation unit 123 allows generating an electric field which performs both tasks of releasing the fluid sample 100 from the immobilized immobilizer 102 and separating the released fluid sample 100. Thus, the electric field generation unit 123 when operated as described above also serves as a release unit 117 for releasing the fluid sample 100 from the immobilized immobilizer 102. The direction of motion of the released fluid sample 100 during the separation is indicated by reference numeral 183. Thus, the released fluid sample 100 can then be separated into fractions of different sizes and charges according to the principle of electrophoresis. The individual fractions can be detected in the detector 50, e.g. optically.

[0105] Figure 2A A three-dimensional view of a portion of a liquid sample separation device 10 according to some embodiments of the present application is shown, demonstrating the application of a separation medium 112 Figure 2 Similar embodiments, but showing some alternatives.

[0106] First, Figure 2A Embodiments of the electrophoresis gel of the separation medium 112 Figure 2A can be applied directly as a strip on a planar support surface or carrier 115. Second, Figure 2A Embodiments are shown in which a second component 110 (e.g. sodium alginate) can have been mixed with the separation medium 112 before applying the separation medium 112 to the support surface or carrier 115. Furthermore, Figure 2AIt is shown that a storage container 111 (such as a needle, a syringe, a pipette, a nozzle, etc.) can inject a fluid sample 100 that has been pre-mixed with a first component 108 (e.g. calcium chloride) directly into the separation medium 112. In particular, the opening of the storage container 111 can be immersed into the medium 112. Alternatively, the opening of the storage container 111 can be spaced apart from the surface of the separation medium 112 and a droplet 119 of the fluid sample 100 pre-mixed with the first component 108 can be jetted by e.g. a printing process. In other words, the fluid sample 100 can be injected directly into the separate or exposed (in particular gel-type and / or liquid) separation medium 112. Due to the addition of the two (or more) ingredients or components 108, 110 of the fixative 102, the immobilization of the fluid sample 100 can take place substantially immediately after the initial contact between the components 108, 110, one of which is pre-mixed with the fluid sample 100. This immediate, temporary and releasable immobilization of the fluid sample 100 upon initial injection into the separation medium 112 can prevent an undesired spatial expansion of the fluid sample 100 in the separation medium 112, thereby ensuring substantially identical starting conditions for different parts of the fluid sample 100 at the beginning of the subsequent separation process of separating the fluid sample 100 into fractions.

[0107] Figure 3 A sample separation device 10 according to one exemplary embodiment of the present application is schematically shown.

[0108] Figure 3 A process flow according to one exemplary embodiment of the present application is shown. After supplying a fluid sample 100 (see block 190), the fluid sample 100 can be subjected to a temporary immobilization by the immobilization unit 116. After the desired waiting time has expired, the fixative 102 can be removed or released from the fixative 102 by disintegration in the disintegration unit 118 and / or by applying a release force by the release unit 117 (in this case, disintegration of the fixative 102 is not necessary, but additionally possible). Thereafter, the fluid sample 100, now enabled to move freely again, can be subjected to an analysis in the analysis unit 120. In the shown embodiment, the analysis unit 120 comprises a separation unit 30 for separating the fluid sample 100 into its fractions. Subsequently, a detection unit or detector 50 of the analysis unit 120 can detect the separated fractions of the fluid sample 100. Optionally, the separated fractions of the fluid sample 100 can be fractionated by a fractionator 60.

[0109] Figure 4A portion of a sample separation device 10 according to an exemplary embodiment of the present application is shown, in which a plurality of gel beads 104 with immobilized (i.e. confined within an immobilizing fixative 102) fluid samples 100 are sequentially injected from a supply channel 167 into a separation channel 169 for subsequent separation of the fluid samples 100 into fractions.

[0110] An injection unit 40 (only schematically shown in Figure 4 as arrows indicating the direction of flow of the beads 104 and fluid samples 100, respectively) is provided for injecting the fluid samples 100 temporarily immobilized within the beads 104 and subsequently released into the separation channel 169. Thus, the injection unit 40 is used for injecting the immobilized fluid samples 100 into the separation channel 169 to direct the fluid samples 100 to the sample separation unit 30. The separation channel 169 can be a channel or fluid conduit in which the fluid samples 100 are to be separated, or can be a separation channel 169 for directing the fluid samples 100 to the sample separation unit 30 (not shown in Figure 4 It can be seen from Figure 4 that the sequence of beads 104 can be driven along the supply channel 167 in an immobilized state. In a transition region 163 at the fluidic interface between the supply channel 167 and the separation channel 169, the fluid samples 100 can be released from the immobilized state (e.g. by changing the chemical environment to disintegrate the fixative 102, by increasing the temperature to liquefy the previously gelatinous or solid fixative 102, and / or by applying an electric field such that the fluid samples 100 migrate or diffuse out of the matrix of the still solid or gelatinous fixative 102). Thus, individual beads 104 can wait in the supply channel 167 until the separation device 10 is ready to separate (in particular by electrophoresis) the fluid samples 100 from the respective next bead 104 in or downstream of the separation channel 169. During this waiting time, the immobilized fluid samples 100 are protected from undesired spatial expansion. By taking this measure, the precision of the separation process can be improved.

[0111] For example, the supply channel 167 can transport the fluid samples 100 within the fixative 102 from a first sample separation device (e.g. a liquid chromatography sample separation device, such as an HPLC, Figure 4 (not shown in Figure 4The fluid sample 100 is released from the immobilized immobilizer 102 and thus from the bead 104 can also be achieved by changing the medium surrounding the bead 104 in the supply channel 167 and in the separation channel 169. The medium 112 in the supply channel 167 can be chosen such that the bead 104 remains in a gel phase in the supply channel 167. Furthermore, the medium 112' in the separation channel 163 can be chosen such that the bead 104 disintegrates or transforms into a liquid phase, thereby releasing the fluid sample 100 from the matrix of the immobilized immobilizer 102.

[0112] Figure 5 Two droplets of a fluid sample injected into the channel 200 without spatial focusing are shown. The injection appears wider than the other injection, indicating a broad spatial spreading range and thus poor reproducibility. The injection at the right end of the channel 200 shows a spatially spread or smeared fluid sample 100". Thus, Figure 5 The phenomena the exemplary embodiments of the present application are intended to overcome are shown.

[0113] More precisely, Figure 5 Two droplets of a DNA sample (30 nucleotides; fluorescently labeled with Rh6G) injected into the separation medium 112' in a non-contacting manner are shown with reference numerals 100', 100". Figure 5 The dashed lines in correspond to a channel-like structure of the separation medium 112' with a planar support surface or carrier. The distance between the dashed lines is in the range between 500 pm and 600 pm, for example. The dye-labeled sample droplets are denoted with reference numerals 100', 100", which have been excited by a laser and have been detected by a camera filter setting. Due to the flow within the tubing, the droplet on the right-hand side (see reference numeral 100") is spatially spread compared to the droplet on the left-hand side (see reference numeral 100'). The droplet on the left-hand side is spatially focused. The fluid samples 100', 100" are spread (in the sense of a deviation from an ideal droplet shape or diameter) due to the diffusion and the kinetic impact of the respective droplet 102 upon penetration of the separation medium 112.

[0114] Figure 6 A separation medium 112 with a spatially focused fluid sample 100 according to one exemplary embodiment of the present application is shown.

[0115] In Figure 6 In, the gel portion is denoted with a dashed box and reference numeral 189. The non-gel portion is denoted with reference numeral 191. The gel portion is Figure 6The separation medium 112 is mixed with 0.54% (w / w) sodium alginate (108). Fluid sample 100 has been mixed with a second component 110 of fixative 102 (which is calcium chloride (100 mM)). Following non-contact metering of a sample droplet (see reference numeral 100) in the channel, sodium-calcium ion exchange occurs, resulting in the formation of a hydrogel. The spatial extent of the formed alginate-hydrogel portion is, for example, about 1 mm. Figure 6 On the right-hand side image, fluid sample 100 can be clearly separated from the hydrogel. Therefore, the injection width is ideally the same as the maximum diameter of the metering droplet.

[0116] Figure 7 A space-focused fluid sample 100 at a later time point (i.e., 20 minutes later) during space focusing is shown according to an exemplary embodiment of the invention.

[0117] Figure 7 It shows the relationship with Figure 5 The corresponding setup, but now an implementation of the concept of temporarily immobilizing the fluid sample 100 by means of a fixative 102. According to... Figure 7 Fluid sample 100 has been mixed with calcium chloride (CaCl2), and separation medium 112 has been mixed with sodium alginate. The two injection droplets of fluid sample 100 are highly focused and less... Figure 5 It expands in that way. Figure 7 The dashed lines again represent the channel-like structure of the separation medium 112 on the planar substrate 115. The distance between the two dashed lines corresponds to approximately 500 μm to 600 μm. Even after a 20-minute waiting period, the size of the injection droplet only increases slightly due to the effect of diffusion.

[0118] Figure 8 The separation of a spatially focused fluid sample 100 according to an exemplary embodiment of the present invention is shown.

[0119] for Figure 8 In this implementation scheme, a mixture of two single-stranded DNA fragments (30 and 56 nucleotides, fluorescently labeled: Rhodamine 6G) was mixed with 50 mM CaCl2 and injected into separation medium 112 mixed with 0.54% (w / w) sodium alginate. These media were already present in an open microfluidic system and separation was performed at 50 V / cm. The non-contact metering injection point was highly focused at the beginning and had already separated relative to the separation medium through the hydrogel barrier. By applying voltage, biomolecules can actively move through the hydrogel barrier and can be injected into the separation medium. After a migration time of 2 minutes, clear separation of the two fragments was observed, indicating that the method is working (compare with...). Figure 8 ).

[0120] Figure 8 A fluid sample 100 is shown that was non-contact injected in a microfluidic electrophoresis system using a droplet printer. The sample contained two single stranded DNA fragments (30 and 56 nucleotides, label: Rhodamine 6G) and had been mixed with 50 mM calcium chloride. The separation medium that established the open microfluidic system had been mixed with 0.54% (w / w) sodium alginate. Images were captured during separation at 50 V / cm at different time points Figure 8 . The images have been aligned based on the smaller fragment. After 2 minutes of migration, the separation of the two fragments is clearly visible and indicates that the described method is working properly.

[0121] It should be noted that the term "comprising" or "including" does not exclude other elements or features and the "a" or "an" does not exclude a plurality. Elements or features described in association with different embodiments can be combined. It should also be noted that reference signs in the claims should not be construed as limiting the scope of the claims.

Claims

1. A method of processing a fluid sample (100) in a sample separation device (10), wherein the method comprises processing the fluid sample (100) in the sample separation device (10) in the following way: immobilizing the fluid sample (100) at least partially by a fixative (102) to suppress spatial expansion of the fluid sample (100) during a waiting time before separation, the method comprising triggering an immobilization phase transition of the fixative (102) to immobilize the fluid sample (100), the immobilization phase transition being from one of the group consisting of a liquid phase, a gas phase and a low viscosity phase to one of the group consisting of a solid phase, a gel phase and a high viscosity phase; subsequently releasing the fluid sample (100) at least partially from the fixative (102); and separating the fluid sample (100) after the release, and wherein the immobilization process is one or more times reversible.

2. The method of claim 1, comprising the following features at least one of: wherein the method comprises triggering the release by applying a release force to trigger migration of at least part of the fluid sample (100) out of the immobilized fixative (102); wherein the method comprises triggering the release by at least partially disintegrating the fixative (102); wherein the method comprises releasing the fluid sample (100) at least partially from the fixative (102) by waiting for a spontaneous disintegration of the fixative (102); wherein the immobilization comprises embedding at least part of the fluid sample (100) into the fixative (102) and / or enclosing at least part of the fluid sample (100) by the fixative (102).

3. The method according to claim 2, wherein the release force is an electrical release force.

4. The method according to claim 2, wherein the migration is diffusion.

5. The method according to claim 2, wherein triggering migration of at least part of the fluid sample (100) out of the immobilized fixative (102) is triggering migration of at least part of the fluid sample (100) through the immobilized fixative (102).

6. The method according to claim 2, wherein the release is triggered by at least partially disintegrating the fixative (102) thermally, electrically and / or chemically.

7. The method according to claim 2, wherein the spontaneous disintegration of the fixative (102) is passively waited for.

8. The method according to claim 2, wherein the method comprises triggering a disintegration phase transition of the fixative (102).

9. The method according to claim 8, wherein triggering a disintegration phase transition of the fixative (102) is a disintegration phase transition from one of the group consisting of a solid phase, a gel phase and a high viscosity phase to one of the group consisting of a liquid phase, a gas phase and a low viscosity phase.

10. The method according to claim 2, wherein disintegrating the fixative (102) is opposite to the immobilization phase transition of the fixative (102) used to immobilize the fluid sample (100).

11. The method according to claim 8 or 9, wherein the disintegration phase transition of the immobilization agent (102) is opposite to the immobilization phase transition of the immobilization agent (102) used for immobilization of the fluid sample (100).

12. The method according to any one of claims 1 to 8, comprising at least one of the following features: wherein the method comprises: forming at least one bead (104) comprising the fluid sample (100) and the immobilization agent (102) upon immobilization of the fluid sample (100); wherein the method comprises shielding the immobilized fluid sample (100) from a medium (106) by immobilizing the fluid sample (100) in an environment of the immobilization agent (102); wherein the fluid sample (100) is a biological sample.

13. The method according to claim 12, wherein the medium (106) is a fluid medium and / or a gel medium.

14. The method according to claim 12, wherein the method comprises contacting the fluid sample (100) with the medium (106) by releasing the fluid sample from the immobilization agent (102).

15. The method according to any one of claims 1 to 8, wherein the immobilization agent (102) comprises a first component (108) and a second component (110), the first and second components being configured such that the immobilization agent (102) is immobilized by an interaction between the first component (108) and the second component (110).

16. The method according to claim 15, wherein the interaction is a reaction.

17. The method according to claim 15, comprising at least one of the following features: wherein the first component (108) is calcium chloride or calcium lactobionate and the second component (110) is sodium alginate; wherein the interaction triggering immobilization is an ion exchange between the first component (108) and the second component (110); wherein the interaction triggering immobilization is a hydrogel formation involving the first component (108) and the second component (110); wherein the first component (108) and the second component (110) are thrombin and fibrinogen; wherein the first component (108) and the second component (110) are polymerizable substances and a crosslinker; wherein the method comprises triggering immobilization by mixing the fluid sample (100) with the first component (108) and then injecting the mixture into a medium (106, 112) comprising the second component (110), wherein the medium (106, 112) comprises one of the group consisting of a gel and a mobile phase; wherein the method comprises providing a separation medium (112) for separating the fluid sample (100) on a carrier (115), supplying the fluid sample (100) to the separation medium (112) thereby triggering the immobilization, and separating the fluid sample (100) by the separation medium (112) after the immobilization and the release; wherein the method comprises: local immobilization of the fluid sample (100) by the immobilization agent (102) while the environment of the fluid sample (100) and the immobilization agent (102) is kept in a fluid state; wherein the method comprises: preparation of the fluid sample (100) for subsequent separation after the immobilization, and performing a separation of the fluid sample (100) after the preparation and after the release; wherein the at least partial immobilization of the fluid sample (100) is performed before and / or after the separation of the fluid sample; wherein the method comprises: applying the fluid sample (100) on the carrier (115), and subsequently applying a separation medium (112) on the carrier (115) and for interaction with the fluid sample (100).

18. The method according to claim 17, wherein the gel is an electrophoretic gel.

19. The method according to claim 17, wherein the mobile phase is a chromatographic mobile phase.

20. The method of claim 17, wherein, providing a separation medium (112) for separating the fluid sample (100) on a carrier (115), supplying the fluid sample (100) to the separation medium (112) thereby triggering the immobilization, and separating the fluid sample (100) by the separation medium (112) after the immobilization and the release, and wherein separating the fluid sample (100) by the separation medium (112) refers to separating the fluid sample (100) by electrophoresis.

21. A sample separation device (10) for separating a fluid sample (100), wherein the device (10) comprises an immobilization unit (116) configured to at least partially immobilize the fluid sample (100) by an immobilization agent (102) to suppress spatial expansion of the fluid sample (100) within a waiting time until separation, the immobilization unit (116) being configured to trigger an immobilization phase transition of the immobilization agent (102) to immobilize the fluid sample (100), the immobilization phase transition being from one of the group consisting of a liquid phase, a gas phase and a low viscosity phase to one of the group consisting of a solid phase, a gel phase and a high viscosity phase; and the device (10) comprises at least one of a release unit (117) and a disintegration unit (118): wherein the release unit (117) is configured as a release unit (117) for at least partially releasing the fluid sample (100) from the immobilization agent (102); wherein the disintegration unit (118) is configured for triggering at least partial disintegration of the immobilization agent (102) after the immobilization to release the fluid sample (100), and wherein, the immobilization process is one or more times reversible.

22. The device (10) according to claim 21, wherein the release unit (117) is configured for releasing the fluid sample (100) from the immobilization agent (102) by applying an electric field.

23. The device (10) according to claim 21 or 22, comprising at least one of the following features: wherein the device (10) comprises a fluid driver (20) configured for driving the fluid sample (100) in a mobile phase towards a sample separation unit (30) after the release, the sample separation unit being configured for separating the fluid sample (100); wherein the device (10) comprises an electric field generation unit (123) configured for generating an electric field for applying an electric force to the fluid sample (100) in the medium (112) during and / or after the release for releasing the fluid sample (100) from the immobilized fixative (102) and / or for separating and moving the fluid sample (100) towards a detector (50) for detecting separated fractions of the fluid sample (100); wherein the device (10) comprises an injection unit (40) for injecting at least a partially immobilized fluid sample (100) into a separation channel (169); wherein the device (10) comprises a compensation unit (121) configured for computationally at least partially compensating for a deviation between a separation result in the presence of the fixative (102) and a separation result in the absence of the fixative (102); wherein the device (10) is a microfluidic device; wherein the device (10) comprises a detector (50) configured to detect a separated fraction of the fluid sample (100); wherein the device (10) comprises a fractionation unit (60) configured to collect a separated fraction of the fluid sample (100); wherein the device (10) comprises a degassing device (27) for degassing a mobile phase; wherein the device (10) comprises an injection unit (40) for injecting the fluid sample (100) into a mobile phase in a flow path between the fluid driver (20) and the sample separation unit (30); wherein the device (10) is configured as an electrophoretic sample separation device or a chromatographic sample separation device.

24. The device (10) according to claim 23, wherein at least a partially immobilized fluid sample (100) is injected into a separation channel (169) in which the fluid sample (100) is to be separated or into a separation channel (169) for guiding the fluid sample (100) to a sample separation unit (30).

25. The device (10) according to claim 23, wherein the electrophoretic sample separation device is a gel electrophoretic sample separation device.

26. The device (10) according to claim 23, wherein the chromatographic sample separation device is a liquid chromatographic sample separation device, a gas chromatographic sample separation device or a supercritical fluid chromatographic sample separation device.

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