EP3784140A1

EP3784140A1 - Device and method for preparing sample material

Info

Publication number: EP3784140A1
Application number: EP19719508.4A
Authority: EP
Inventors: Franz Laermer; Jochen Hoffmann; Tino Frank; Paul Kallenberger
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2018-04-26
Filing date: 2019-04-24
Publication date: 2021-03-03
Also published as: WO2019206944A1; CN111989048A; US20210114024A1; DE102018206469A1

Abstract

The invention relates to a device (1) for preparing sample material. The device (1) is designed as a rotary device by means of which a defined quantity of liquid can be drawn in into a sample receiving chamber (15) for the sample material or can be expelled from the sample receiving chamber (15) for the sample material, by means of a rotary motion.

Description

description

title

Apparatus and method for preparing sample material

The invention relates to an apparatus and a method for preparing sample material. The invention also relates to a microfluidic system with such a device.

State of the art

From the German patent application DE 10 2005 050 347 Al is a

Sampling device, in particular a biopsy needle, known, consisting of a hollow needle with a distal opening with a

Peripheral edge and guided in the hollow needle slidable stylet with a tip and a length that the tip of the distal opening of the hollow needle can protrude. The sampling device is designed, for example, as a fine needle biopsy device. The sampling device or fine needle biopsy device is used for the removal of animal, human and / or plant tissue. through

Fine needle biopsies are taken in suspected cases tissue material or cells from, for example, lung, thyroid or prostate. Classically, this sample material is applied to a slide and examined by a pathologist. Here, for example, the morphology of the cells is optically examined. The so-called immunohistochemical staining identifies cell-specific features. In addition, more and more genetic characteristics of the cells are determined. The necessary sample preparation steps are usually extensive and are therefore often not carried out promptly. This sometimes results in therapies being prescribed without the knowledge of relevant mutational status. Disclosure of the invention

The device for preparing sample material is advantageous as

Turning carried out, with the rotational movement of a defined amount of liquid can be drawn into a sample receiving space for the sample material or ejected from the sample receiving space for the sample material. The sample receiving space preferably has a microfluidic volume, in particular a liquid volume, of one to one thousand microliters, preferably between ten and one hundred microliters. The sample material is, for example, animal, human and / or plant tissue. The sample material is taken, for example, with a suitable sampling device. In the

Sampling device is for example a biopsy needle or a biodetector with a functional or functionalized surface. The functional or functionalized surface advantageously serves to isolate molecules or cells from the human body. With the biopsy needle, a patient, cells, especially tumor cells, can be removed, which circulate in the bloodstream. The functional or functionalized part of the biopsy needle is advantageously coated so that either cell-free DNA or cells of epithelial origin which express a certain surface protein, for example EpCAM, upon contact with the needle surface are bound by antibodies present there, for example anti-Ep-CAM , EpCAM is an abbreviation for the English term Epithelial Cell Adhesion Molecule. For example, in one application, the biopsy needle or biodetector is placed in the arm vein of a patient for thirty minutes, removed and washed. A physician then determines the number of fixed cells and / or determines the mutation status of the cells. In a microfluidic system, small sample volumes can be used with a high volume

Sensitivity are analyzed. Automation, miniaturization and parallelization also allow a reduction of manual steps, as well as a

Reduction of errors caused thereby. However, prior to a microfluidic analysis process, the macroscopic sample must first be transferred to the microscopic or fluidic fluid environment. This so-called world-to-chip interface requires the standard

Preparation of an input solution or input sample.

Typically, the sample preparation is performed off-chip - manually or with another device. As a result, by fluid transport between different vessels and the use of different

Solvents and washing steps, which is diluted even more deeply concentrated sample. There is also the risk of contamination or decomposition of the fragile sample material by time-consuming steps.

A point-of-care analysis that most lab-on-chip applications require requires rapid sample analysis, without complicated and labor-intensive operations, which are usually performed only by trained personnel in central laboratories.

Microfluidic systems are often needed for various applications. A universal network system allows the analysis of

different problems. The difference between the different approaches is often a different type of sample preparation. For this, the microfluidic analysis unit would often have to be readjusted, which involves a high development effort. Thus, for example, if a sample of a needle biopsy is to be transferred to a fluid sample system, a solution must be found for pre-processing a rigid needle without the need to change the microfluidic analyzer at high cost (e.g., new injection molded parts). Otherwise, the advantage of almost completely automated processing is lost.

With the claimed rotating device, it is possible quickly, easily and accurately raise well-defined small amounts of different liquids and eject again to microfluidically immobilized samples in the appropriate input form. Thus, an immobilized sample can be converted into a suitable solution, suspension or dispersion. For this, among other things, it must be rinsed, but also the sample material must be peeled off or cell material released by lysis. This small sample volume can then be transferred directly into a microfluidic system for further processing. The claimed rotary device is preferably airtight with a

Connected to cannula-shaped volume. In this volume, the sample is submitted. By a predefined rotation, a defined amount of liquid can be injected and ejected into the cannula volume. This system has the following advantages: The device has an intuitive and simple user design. This also does not allow specially trained personnel handling. In addition, this minimizes the risk of possible errors that could be committed by the user. Volumes are predefined and can not be changed and are pure through the complete

Rotary action defined. In addition, the use of transparent cannulas, for example made of glass, allows an optical visual inspection of the handling processes, which offers additional handling safety. By using transparent cannulas, for example glass, it is furthermore advantageous to carry out an optical analysis of the sample material, for example a

Cell counting with the help of a microscope, allowing the

Sample material is located in a protected environment (inside the cannula). The system is designed with the use of a cannula so that it can be used without loss or dilution. Rigid shapes, such as a swab, wire, needle, or fine biopsy punch, can be inserted into the cannula without changing their geometry. Also, the geometry of the microfluidic analysis system does not have to be specially adapted. This allows existing microfluidic systems to be described herein

To complement the sample preparation system without adapting the system. The system described is largely closed. This will do that

Contamination risk lowered by several factors. The system described can be realized by an ensemble of a few, inexpensive disposable parts. This allows one in the medical field and desired

standard disposable application. Solutions for cleaning and preventing cross-contamination need not be developed. By drawing in and expelling the defined amount of liquid can be the

Reduce the chance of air bubbles inside the sample fluid. For a subsequent analysis, for example one

Microfluidic platform, it is much easier to transfer a liquid sample bubble-free on the chip, as a rigid sample bubble-free on the microfluidic platform in liquid convict. For example, the chemicals needed for washing or lysis could be pre-stored on a chip. This increases the user-friendliness, since the number of individual parts of an overall system is minimized. The application of the described invention is based on the target users known concepts. The operation of tools that have similar functions, such as syringes or pipettes, the patient-oriented personnel (for example, nurses, doctors, paramedics) are very familiar and needs no further training in the

Theme. The system can be built by combining standard luer parts, which are standard in medical fluidics. These parts are standardized and can be adapted by slight modifications to commercially available components (for example, integration of seal, stop point). If samples can not be analyzed on-site, the device allows a first simple one

Sample preparation, which converts the analyte in a small volume into a stable form (for example, fixed DNA) and then can be shipped in and with the small volume adapter. The device can be further used to remove a small volume from a microfluidic system and apply it for further analysis. This is of interest insofar as results are further evaluated in clinical trials. Thus, a special result can be prepared directly for sequencing.

A preferred embodiment of the device is characterized

characterized in that the sample receiving space comprises a volume that is less than ten milliliters. The sample receiving space, for example, has a volume of about ten to two hundred microliters.

A further preferred embodiment of the device is characterized in that the rotating device comprises a rotary body which is threadedly coupled to a fluid receiving space having a volume whose size is changed by rotating the rotary body relative to the thread or by twisting the thread relative to the rotary body becomes. The volume of the liquid receiving space is larger, preferably at least two to three times as large as the volume of

Sample receiving chamber. The rotary body advantageously comprises a radially outward External thread, which is complementary to an internal thread of the aforementioned thread. In addition, the rotary body is advantageously provided with a central through hole which fluidly connects the liquid receiving space to the sample receiving space. The rotary body is designed according to a further embodiment as a Luer-lock element. Depending on the direction of rotation of the relative rotational movement, the volume of the liquid receiving space is smaller or larger. When the volume of the liquid receiving space becomes smaller, then fluid, in particular liquid, is released from the

Fluid receiving space ejected through the sample receiving space.

As the volume of the fluid receiving space increases, fluid is drawn through the sample receiving space into the fluid receiving space. This allows the sample material in a simple way

Preparatory steps, such as washing, fixing and / or lysing, be subjected. As soon as the prepared steps have been completed, the sample, which is now in liquid form, can be transferred in a simple manner, in particular reproducibly, by turning the rotary body. The liquid sample is ejected from the sample receiving space. Thus, the sample can advantageously be transferred directly to a lab-on-chip. Depending on the version, a liquid used for washing, fixing or lysing can already be stored in the lab-on-chip. With the claimed

Device, the required fluids in a corresponding microfluidic system can also be removed from the lab-on-chip. With respect to this embodiment, when the rotary body is rotated, the thread is advantageously fixedly arranged. If the thread is twisted, then advantageously the rotating body is arranged stationary.

Of course, the two parts, so the rotary body and the thread, both are rotated relative to each other. In this context, a thread is referred to in particular as an internally threaded section which meshes with an externally threaded section, which in turn is formed on the rotary body. The female threaded portion is formed, for example, in a hollow body in which the rotary body is rotatable.

A further preferred embodiment of the device is characterized in that the liquid receiving space is bounded by a hollow body which is internally threaded. The hollow body has for example, the shape of a straight circular cylinder jacket closed at one end. In the hollow body of the rotary body via the thread is rotatable so that the limited volume of the hollow body of the

Fluid receiving space changed when the rotary body is rotated in the hollow body. The closed end of the hollow body can be designed as a Luer lock element. Due to the luer-lock element can advantageously a non-functional or functionalized portion of a biopsy needle from the

Fluid receiving space are led out. The biopsy needle is advantageous in its functional or functionalized section

Sample receiving room arranged. Depending on the design of the biopsy needle, the biopsy needle can extend through the fluid receiving space through the closed end of the hollow body, which is advantageously designed as a luer lock element out. Of course, the hollow body can be rotated relative to the rotary body to the volume of

Fluid receiving space defined to reduce or enlarge. Then, the rotary body can be held, for example, with one hand, while the hollow body is rotated with the other hand.

A further preferred exemplary embodiment of the device is characterized in that the hollow body has a sealed push-through area at an end facing away from the sample receiving space. The hollow body has, for example, the shape of a straight circular cylinder jacket which is closed at one end. In the hollow body, the rotary body is rotatable via the thread, so that the volume of the liquid receiving space bounded by the hollow body changes when the rotary body is rotated in the hollow body. The closed end of the hollow body can be designed as a Luer lock element. Due to the luer-lock element can advantageously a non-functional or functionalized portion of a biopsy needle from the

Sample receiving room arranged. Depending on the design of the biopsy needle, the biopsy needle can extend through the fluid receiving space through the closed end of the hollow body, which is advantageously designed as a luer lock element out. A further preferred embodiment of the device is characterized in that a biopsy needle, as shown in Figure 14, in a

Lysis device is introduced. However, this does not have a rotating device for receiving liquid, but a attached, for example, in place 153 Peläusball (or the like). By means of this ball process liquids can be introduced or expelled into the capillary 15 by volume enlargement or reduction. This simplifies the

Fluidaktuierungseinheit over the aforementioned rotation mechanism.

A further preferred embodiment of the device is characterized in that the liquid receiving space fluidly with the

Sample receiving room communicates. As a result, it is achieved in a simple manner that the sample receiving space in the liquid

Liquid receiving space can be fed. At the same time, liquid can be expelled from the liquid receiving space through the sample receiving space.

A further preferred embodiment of the device is characterized in that the sample receiving space is bounded by a tubular body which is open at its end remote from the liquid receiving space. Liquid can be drawn in or expelled through the open end of the tubular body. The tubular body is designed to be similar to a cannula or capillary.

A further preferred embodiment of the device is characterized in that the tubular body or the rotary body a

Having externally threaded portion which is complementary to a

Inside threaded portion is formed, which is arranged in the liquid receiving space. The tubular body is advantageously combined with the rotary body. The tubular body may be integrally connected to the rotary body. Of the

Male thread portion is advantageously formed on a collar which is angled from the tubular body or a main body of the rotary body. The tube body may also have an open end with a tip that is designed to be more or less similar to a pipette tip. Then, the rotary body, for example a cannula or a capillary, with a suitable Sealing device can be fluidly connected to the rotary body in a simple manner.

A further preferred embodiment of the device is characterized in that the tubular body, the hollow body and / or the rotary body are combined with at least one sealing device / is. Thus, the sample receiving space and the liquid receiving space, apart from the open end of the tubular body, fluid-tight, in particular airtight, sealed to the environment.

A further preferred embodiment of the device is characterized in that the tubular body, the hollow body and / or the rotary body are combined with a filter device. With the filter device, the implementation of individual process steps, such as a purification or

Pre-cleaning the sample material to be made more effective.

A further preferred embodiment of the device is characterized in that the liquid receiving space or the

Sample receiving space has a connection for supplying and / or discharging a fluid. The connection is provided, for example, on a connection body of the rotating device. But the connection can also be provided on one or the hollow body, the

Fluid receiving space limited. However, the connection can also be provided on an additional part, for example, as will be described below, on a T-piece. The port is advantageous for supplying a fluid to remove all sample material from the fluid receiving space and the sample receiving space. This advantageously provides a device for the lossless, microfluidic preparation of immobilized sample material, in particular on rigid needles, preferably by means of a two-phase system. Via the connection, a fluid, in particular an oil phase, can be supplied in order to completely remove a sample liquid from the sample receiving space, for example from a cannula, and transfer it lossless into a lab-on-chip platform. The sample liquid is a liquid containing sample material. In addition, over the connection subtasks of a Liquid Vorverlagerung or -vorlegung and the provision of a liquid are taken.

A further preferred embodiment of the device is characterized in that the connection for supplying and / or discharging the fluid is provided as a third connection to a T-piece, the

Limited fluid receiving space and / or the sample receiving space. The third connection is advantageously closed by a closure body. During operation of the device, the closure body advantageously serves to tightly close the third connection, specifically in particular when no fluid is supplied or removed via the third connection. The closure body is advantageously designed as a luer lock element. The T-piece with the third connection is advantageously also designed as a Luer-lock element. The design as a Luer-lock element or Luer-Lock elements, the handling of the device is simplified.

A further preferred embodiment of the device is characterized in that the T-piece has a first connection for the

Having a rotating device, a second connection for the sample receiving space and the third port for supplying and / or discharging the fluid. The third connection can be arranged transversely to the first two connections with respect to the T-piece. The third connection may be arranged in relation to the T piece, but also parallel to the first two connections. A syringe with a Luer connection can be screwed to the third connection of the T-piece or T-element instead of a closure body. Fluid, in particular oil, can be applied in a simple manner via the syringe by actuating a piston of the syringe. The syringe may be attached to the third port of the tee orthogonally but also parallel to a capillary or cannula.

The invention further relates to a microfluidic system with a previously described apparatus for preparing sample material. The microfluidic system includes in addition to the previously described

Apparatus for preparing sample material at least one

microfluidic chip. This is a simple way one fully automated analysis of biological samples on site at the point of care. Reagents are advantageously presented in the fluidic chip. With the reagents, the sample material can then be prepared, for example, washed. The microfluidic chip advantageously comprises a sample input area. The sample input area on the microfluidic chip includes, for example, an input channel adapted to receive the open end of the tubular body of the device described above. The shape of the input channel is advantageously adapted for this purpose to the shape of the tubular body at its open end. The input channel is in the microfluidic chip at at least one junction with a microfluidic

Network connected. As a result, the preparation or processing of the sample material, for example, the washing and

Staining the sample material, considerably simplified.

If appropriate, the invention also relates to a tubular body, a hollow body, a rotary body, a sealing device and / or a filter device for a previously described device for preparing sample material or for a previously described microfluidic system. The parts mentioned are separately tradable. The said parts are advantageously produced inexpensively from a suitable plastic material by injection molding. Depending on the number of parts required, production by three-dimensional printing or rapid prototyping is also possible. Depending on the type of used

Materials are also used in machining processes, such as milling. Individual parts or subregions can also by a

Forming processes, such as hot stamping, are presented. According to a further aspect of the invention, as far as possible, commercially available Luer parts, in particular with the use of a sealing ring, are used. Suitable materials are, for example, biocompatible plastics. The materials used advantageously have a small thermal expansion coefficient. The tubular body is advantageously made of the same material as a capillary, in particular a glass capillary. The tubular body may also be formed of metal. Depending on the design, a conventional syringe cannula can be used as a tubular body. If appropriate, the invention also relates to a method for preparing sample material with a previously described device, in particular in a microfluidic system.

Using biopsy needles, cells from tissues or fluids (blood,

Lymph) can be selectively removed from the patient. These are then lysed in a small volume. Especially in liquid biopsy applications, only a few cells can adhere to the needle by antibody selection and a small volume is of great importance for sensitive analysis. Should cells be transferred from a biopsy needle to a microfluidic platform, the cells adhering to the needle should be lysed in as small a volume as possible. This can be done, inter alia, in a glass cannula. In this case, defined volumes of lysis buffer can be raised. The challenge is often complete

Draining these needles, especially if rare cells (for example tumor cells with specific point mutation, immune cells with special antigen, stem cells) are sought. Any small residue can mean a total loss of information.

The core of the method or the device is the loss-free processing of a limited sample volume in a cannula. The limited sample volume is drawn in by means of a rotary device and ejected again. Possible fluid residues are completely displaced from the cannula via a side channel through an oil phase and the whole

Sample volume can be processed lossless. The oil phase can be used in addition to further processing on a lab-on-chip system.

The application of the device described here and the processes used have the following advantages:

a) A sample volume can be completely removed from a cannula. This is especially an advantage when working with small volumes, little sample material is present (for example, small copy numbers of DNA strands with rare mutational patterns) or the volume for the

Quantification must be exact. b) The oil phase used can be used for subsequent processes. This is an advantage in particular for further processing on a lab-on-chip platform which uses two-phase systems.

c) By feeding oil through the cannula when transferring the sample to a Lab-On-Chip, the sample can be plugged directly into multiple oil phases as a plug and processed directly loss-free on the Lab-On-Chip platform.

d) Volume retention can be achieved by pre-storage on a lab-on-chip platform

be guaranteed. The lysis unit does not have to guarantee exact fitting. The volume retention is guaranteed only by the volumes of the Lab-On-Chip platform.

e) The invention can be made of commercially available, standardized Luer parts

realize. In particular, these parts are made of bio- and PCR-compatible materials. PCR means polymerase chain reaction.

f) The device also makes it possible to drive a multi-stage lysis process lossless.

g) The fluid method which enables the device reduces the

manual steps, which take place before the automatic on-chip processing, to a minimum. Also, these do not have to be metric-precise. The precision is guaranteed by the volume shift.

h) Complete, lossless evacuation avoids readjustment of volumes and possible dilutions. Readjusting always means that a volume measurement and a feedback system must be implemented. This leads to more complex systems and more development effort, which is limited in point-of-care applications.

Suitable materials are biocompatible plastics. Obviously, the same material from which the cartridge or a container of the

microfluidic system is used. The material should have a small thermal expansion coefficient. As already mentioned, a glass capillary or even a syringe cannula made of metal can be used for the capillary. Standard luer parts can also be used.

Syringes can be made of plastic or glass, but should be sealed (by Luer connection or gluing) connected to the T-element. Further advantages, features and details of the invention will become apparent from the following description in which, with reference to the drawings, various embodiments are described in detail.

Short description of the drawing

Show it:

Figure 1 is a schematic representation of an apparatus for preparing sample material with a rotary body which is rotatably arranged in a hollow body, in longitudinal section;

Figure 2 is a schematic representation of a tubular body, which is connected by means of a sealing device fluid-tight with a coupling body, in

Longitudinal section;

Figure 3 is a schematic representation of a tubular body, with a

Connecting body is combined, in longitudinal section;

Figure 4 shows a device similar to that in Figure 1 with a sealing device and with two stops in longitudinal section;

Figure 5 is a similar device as in Figure 4 with a

Sampling device in longitudinal section;

Figure 6 shows a similar device as in Figure 5 with a symbolic

indicated PCR bead on the sampling device;

Figure 7 shows a similar device as in Figure 5 with an additional third stop in longitudinal section;

Figure 8 shows the device of Figure 7 after passing over the additional stop in a longitudinal section; Figure 9 is a flow diagram illustrating a method of preparing sample material with a device such as shown in Figures 1 and 4 through 8;

FIG. 10 shows a microfluidic system with a lab-on chip and an open end of a tubular body of the device from FIG. 1 in section;

11 shows the open end of the tubular body of the device of Figure 1 with sample material, with a microfluidic container and with a

Magnetic device in longitudinal section;

FIG. 12 shows the device from FIG. 5 with an additional filter device at the open end of the tubular body in longitudinal section;

Figure 13 is a similar device as in Figure 1 with a

Sampling device and with a sliding seal in longitudinal section;

FIG. 14 shows a further embodiment of a device for preparing sample material with an additional T-piece; the

Figures 15 to 17 is a schematic illustration of a method of using the apparatus of Figure 14 to completely transfer fluid from a cannula;

Figure 18 shows a variant of the device of Figure 14 when attaching a syringe to the tee;

Figure 19 is a similar view as in Figure 18 when attaching a syringe from the top of the tee; the

Figures 20 to 23 is a schematic representation of a method, such as the device of Figure 14, which can be advantageously integrated into a fluidic flow for a lysis process with adhering to a biopsy needle cells in a lab-on-chip platform; and the Figures 24 to 26 an application of the apparatus of Figure 18, wherein two phases are preceded in the syringe.

Description of the embodiments

1 shows a device 1 designed as a rotating device for preparing sample material 23, 33 (in FIGS. 2, 3, 5 through 8, 12, 13), shown schematically in longitudinal section. The rotating device 1 comprises a rotary body 2, which is rotatable by means of a thread 3 in a hollow body 4. The hollow body 4 has the shape of a straight circular cylinder, which is closed at its upper end in Figure 1. The hollow body 4 limits one

Fluid receiving space 5.

The thread 3 comprises an internally threaded portion 6 in the hollow body 4. In the female threaded portion 6 engages an externally threaded portion 7 of the thread 3 a. The male threaded portion 7 is formed on a collar 8 of the rotary body 2. The collar 8 is angled from a base body 9 of the rotary body 2.

The main body 9 of the rotary body 2 comprises a central through hole, which merges into a tubular body 10. The tubular body 10 is designed, for example, as a capillary 11 with an open bottom end 12. The capillary 11 bounded inside a sample receiving chamber 15 which is fluidly connected via the central through hole in the rotary body 2 with the liquid receiving space 5 in the hollow body 4.

A double arrow 13 indicates in FIG. 1 that the tubular body 10 moves up and down with the rotary body 2 when the rotary body 2 is rotated relative to the hollow body 4. The movement of the rotary body 2, which is indicated by the double arrow 13, is also referred to as stroke. By the stroke of the rotary body 2, the volume of the liquid receiving space 5 changes, as indicated by a double arrow 14 in Figure 1.

When the volume of the liquid accommodating space 5 becomes smaller, liquid is taken from the liquid accommodating space 5 through the liquid Sample receiving chamber 15 and the open end 12 of the tubular body 10 ejected. As the volume of the liquid receiving space 5 becomes larger, liquid is drawn into the liquid receiving space 5 through the open end 12 of the tube body 10. The liquid is provided at the open end 12 of the tubular body 10 via a suitable container (not shown in FIG. 1).

FIG. 2 shows how a tubular body 20, which is designed as a capillary 21, can be connected to a rotary body (2 in FIG. 1). To depict a fluid-tight connection, a seal 22 is provided on the capillary 21

appropriate. Above the seal 22, an end portion of the capillary 21 is disposed within a coupling body 26. The coupling body 26 includes a tip 27 that is made similar to the tip of a pipette. The free end of the tip 27 of the coupling body 26 bears sealingly against the seal 22.

The coupling body 26 is connected, for example, to a rotary body as shown in FIG. 1 and designated 2. The connection between the rotary body and the coupling body 26 may be made in one piece.

In the capillary 21, sample material 23 is arranged on a sampling device 24. The sampling device 24 is designed as a biopsy needle 25. In FIG. 2, only one functional or functionalized section of the biopsy needle 25 is arranged in the capillary 21.

FIG. 3 shows a tubular body 30 designed as a cannula 31. In the cannula 31, sample material 33 is arranged on a sampling device 34. The sampling device 34 comprises, for example, a biopsy needle 35 whose functional section is arranged inside the cannula 31. The rest of the biopsy needle 35 is passed through a connection body 39 with a coupling device 37.

The connecting body 39 is integrally connected to the tubular body 30. On its right-hand side in FIG. 3, the connecting body 39 has a connection 40, for example for a rotating device (not shown in FIG. 3). The connection 40 is combined with a filter device 32. The coupling device 37 comprises a luer lock element 38, which comprises a push-through region for the biopsy needle 35. A free end 36 protrudes in Figure 3 above from the coupling device 37 out.

The connection body 39 tapers toward the tubular body 30. Of the

Connecting body 39 with the tubular body 30 and the coupling device 37 and the filter device 32 is designed for example as a disposable part. The rotary device which is connected to the terminal 40 is then designed, for example, as a reusable part. The filter 32 advantageously serves to reduce the risk of contamination.

FIG. 4 shows how a predefined volume can be drawn in with a turning device 41. For this purpose, the rotating device 41 must be airtight and rotated when drawing in liquid by a precisely defined number of revolutions. The rotating device 41 comprises a

Rotary body 42 which is coupled via a thread 43 with a hollow body 44. A sealing device 45 is arranged for sealing between the rotary body 42 and the hollow body 44.

The thread 43 is provided in the hollow body 44 with two stops 46, 47, by which the rotation of the rotary body 42 is limited in the hollow body 44. The rotary body 42 comprises a main body 49 with a central through hole. Of the main body 49, a collar 48 is angled in Figure 4 above. At its lower end in FIG. 4, the rotary body 42 is connected to a tubular body 50.

The tubular body 50 includes a sample receiving space 15 which is connected to a liquid receiving space 5 within the rotary body 42 and within the hollow body 44, respectively. Due to the enlarging space within the rotating device 41 creates a negative pressure, which ensures that a well-defined volume of liquid is sucked into the rotating device 41 via the open end of the tubular body 50.

In FIGS. 5 and 6, a device 51 designed as a rotary device is shown with a rotary body 52, which is connected via a thread 53 in a Hollow body 54 is rotatable. A sealing device 55 serves to seal between the rotary body 52 and the hollow body 54 with the thread 53. The rotary body 52 comprises a collar 58, which is angled from a base body 59. The main body 59 is connected to a tubular body 60, which is designed as a capillary or cannula.

In the tubular body 60, a sampling device 34 is arranged, which is designed as a biopsy needle 35. The sample material 33 is at the

Sampling device 34 is arranged and is prepared by means of liquid for analysis. The liquid is sucked into the liquid receiving space 5 through the sample receiving space 15 inside the tube body 50.

For this purpose, the rotary body 52 is rotated defined. The rotation of the rotary body 52 relative to the hollow body 54 is limited by two stops 56, 57 on the thread 53.

The rotary body 52 may also be referred to as an adapter piece and is designed in Figure 5 as a Luer-Lock with a Gummispetum. This provides the advantage that a rigid sample, such as the biopsy needle 35, or a functionalized rotary, is already equipped with a Luer lock. This eliminates a complicated processing of the wire, which greatly minimizes the risk of unwanted contamination or destruction of the sample. In addition, the sample does not have to be prepared awkwardly, but can be used directly and treated.

FIG. 6 shows the possibility of a combination of several steps for later realizing a quantitative real-time polymerase chain reaction (PCR) within the device 51. A PCR bead 61 is shown by way of example within the rotary body 52. For example, chemicals needed for preparation, such as lyophysate, may be pre-stored in dried form.

FIGS. 7 and 8 show a device 71 designed as a rotating device with which a multi-step method can be carried out. The device 71 comprises a rotary body 72, which by means of a thread 73 in a hollow body 74 is rotatable. For sealing between the hollow body 74 and the rotary body 72, two sealing devices 75, 76 are provided.

The rotary body 72 comprises a collar 78, which is angled away from a main body 79 of the rotary body 72. The main body 79 of the rotary body 72 passes into a tubular body 80, which, as in the preceding

Embodiments, the sampling device 34 includes.

The hollow body 74 is combined with a sealing cylinder 77. The sealing cylinder 77 has the shape of a straight circular cylinder jacket and is closed at its upper end in Figure 7. A non functional section of the

Sampling device extends through the closed end of the sealing cylinder 77th

The rotary body 72 comprises a central recess 70 in which the lower end of the sealing cylinder 77 engages in FIG. The sealing cylinder 77 is firmly connected to the hollow body 74. The rotary body 72 is relative to the

Seal cylinder 77 rotatable. The thread 73 and the sealing means 75, 76 are arranged in an annular space, which is bounded radially inwardly by the sealing cylinder 77 and radially outwardly from the hollow body 74. The collar 78 of the rotary body 72 is rotatable by the thread 73 between a total of three stops 66, 67, 68.

66 denotes a lower stop. 67 denotes an upper stop. An additional central stop 68 is disposed between the two stops 66 and 67. In Figure 7, the collar 78 of the rotary body 72 between the two stops 66 and 68 is arranged. With the

different stops 66 to 68 can be defined in a simple manner different volumes that are ejected or retracted by the device 71 during rotation of the rotary body 72 relative to the hollow body 74.

When carrying out a multi-step method with the device 71 in FIG. 7, for example, a first volume VI is drawn up once or several times in a first step. For this purpose, the rotating device 71 with the Bund 78 rotated from the lower stop 66 to the additional central stop 68. In a further step is metered with a second volume V2 by being turned up to the upper stop 67. The second volume is larger than the first volume.

In Figure 8 it is shown how the rotary body 72 is moved with the collar 78 between the two stops 66 and 67 to retract or eject the second volume. The additional central stop (68 in Figure 7) is destroyed in Figure 8 after a single run over and thus no longer available.

In FIG. 9, the implementation of a method is represented by rectangles 81 to 84, like the device 1; 41; 51; 71; 131 is used to sample a, for example, cells equipped with sampling device 24; 34, in particular a wire-equipped wire, for genetic analysis to prepare. In this case, the cells are to be lysed in the device and the lysate transferred to a microfluidic analysis unit. By the rectangle 81 a washing step is indicated. The sample needle is washed, for example, with a phosphate-buffered saline solution to remove susceptible residues from the sample receptacle, for example, blood, fat, culture medium. For this purpose, the washing liquid is drawn through the open end of the tubular body in the device with the sample and ejected again.

By the rectangle 82, a step fixing is indicated. When fixing, the sample is biologically fixed so that no more biochemical reactions take place in the cells. For this purpose, a fixing solution, for example

Formaldehyde or acetone, mounted with the device, incubated and ejected again. Subsequently, it is advantageous to briefly wash again.

The rectangle 83 indicates a step of lysing. In lysing, internal cell material, such as proteins or nucleic acids, is lysed

Approved. For this purpose, a lysis solution, for example distilled water, is raised and the cells are incubated therein.

By the rectangle 84, a step sample transfer is indicated. In this case, the lysate from step 83 is transferred directly into a microfluidic analysis unit. The microfluidic analysis unit belongs to a microfluidic system, as indicated by 100 in FIG.

The microfluidic system 100 in FIG. 10 comprises a lab-on-chip 101. The lab-on-chip 101 comprises a microfluidic channel system 102

Sample transfer is the tubular body 10 with its opening 12 in a

Insertion opening 103 of the lab-on chip 101 is arranged. The positioning of the tubular body 10 is facilitated by a stop 104 in the insertion opening 103.

With the aid of the rotating device, a defined amount of fluid, in particular liquid, for preparing a sample from the lab-on-chip 101 can then be drawn in in a simple manner. After preparing the sample, it can then also be ejected by means of the rotating device into a corresponding sample chamber of the lab-on chip 101.

FIG. 11 shows an application in a microfluidic system 110 with a possible magnetic cleaning. The microfluidic system 110 comprises a microfluidic container 111 with magnetic beads 112

Magnetic cleaning, the magnetic beads 112 with a

functionalized surface introduced into the tubular body 10 and ejected again. Depending on the type of functionalization, the beads bind interfering constituents or the sample sought. For this purpose, the tube body 10, which is designed, for example, as a cannula, is advantageously encased with a magnetic device 113. The magnetic beads 112 are transported by a corresponding movement of the magnetic device 113 in the tubular body 10.

FIG. 12 shows, using the example of the device 71 shown in FIG. 7, that a filter device 120 can also be placed at the open end 12 of the tubular body 80. With the filter device 120, a purification or pre-cleaning of the sample can be implemented in a simple manner.

FIG. 13 shows a device 131 which is similar to the device 1 from FIG. To designate the same or similar parts are the same Reference numeral as used in Figure 1. To avoid repetition, reference is made to the preceding description of FIG.

In the device 131 is a sampling device 34 with

Sample material 33 is arranged. In contrast to the device 1 in Figure 1, the device 131 comprises a sliding seal 132 for sealing between the rotary body 2 and the hollow body 4. The sliding seal 132 allows a relatively simple seal and is advantageously attached to the hollow body 4.

FIGS. 14 to 26 show a device 141 for preparing

Sample material with a rotating device 142 shown schematically in various designs and applications. The device 141 is closed at its upper end in the figures by a septum 143 fluid-tight and pressure-tight. The septum 143 is, for example, a plug made of an elastic material, through which a biopsy needle 144 is inserted.

The biopsy needle 144 extends through the septum 143 into the interior of the device 141. In a functional region 145 of the device 141, a section 146 of the biopsy needle 144 is arranged. The portion 146 of the biopsy needle 144 is preferably a functionalized portion.

The rotary device 142 comprises a rotary body 147, which, as described above, via a (not shown in Figures 14 to 26) thread in the axial direction, ie in the figures 14 to 26 downwardly and upwardly, is movable when the Rotary body 147 is rotated.

The functional area 145 of the device 141 is designed as a sample receiving body 148. The sample receiving body 148 may also be referred to as a tubular body and is designed, for example, as a capillary.

Between the sample receiving body 148 and the rotating device 142, the device 141 includes a T-piece 150. The T-piece 150 has a first port 151 for the sample receiving body 148 and a second port 152 for the rotary device 142 on. The biopsy needle 144 extends, as indicated in FIG. 14 by a dashed line 149, lengthwise from top to bottom through the septum 143, through the rotary body 147 and through the T-piece 150 into the functional area 145 of the device 141.

The T-piece 150 comprises a third connection 153, which is closed in FIG. 14 in a fluid-tight and pressure-tight manner by a closing body 155. In FIGS. 14 to 18, 21, 22 and 24 to 26, the third connection 153 of the T-piece 150 is arranged perpendicularly or transversely to the longitudinal extent of the device 141.

In Figure 19, the device 141 is shown with a T-piece 170, in which terminals 171 and 172 correspond to the terminals 151 and 152 of the T-piece 150. A third port 173 is different from the T-piece 150 in the T-piece 170 parallel to the longitudinal extent or

Longitudinal axis of the device 141 is arranged.

The sample receiving body 148 in FIG. 14 is, for example, a cannula in which the functionalized biopsy needle 144 is disposed with sample material for fluidic processing. For this purpose, the

Sample receiving body 148 is connected via the T-piece 150 to the rotating device 142, with which by a rotational movement of the rotary body 147 fluid can be drawn through the open end of the cannula 148 and ejected.

Through the T-piece 150 with the third port 153 a channel

provided that can be used in the operation of the device 141 to convey liquids from above through the T-piece 150 into the cannula 148. Thus, a flow or a flow through the cannula 148 can be generated in a simple manner.

The closure body 155 is designed for example as a rotary lid closure and preferably normalized for Luer parts. If desired, the closure body 155 may be unscrewed to introduce a fluid into the device 141 via the third port 153 of the tee 150 via a suitable device, such as a syringe. FIGS. 15 to 17 show how the device 141 from FIG. 14 is used to completely remove a fluid, in particular a fluid 154 containing the sample, which is also referred to as a sample, from the cannula 148.

For this purpose, as indicated in FIG. 17, an inert phase 159, in particular an oil phase, is transported through the cannula 148 in order to collect the fluid, in particular the sample material 154, in a container 156 at the open end of the cannula 148. The container 156 preferably belongs to a lab-on-chip.

The process illustrated in FIGS. 15 to 17 is particularly in the

Connection with a biopsy needle of importance, if the adhering to the biopsy needle material, usually cells, transferred in the smallest possible volume and the total volume to be processed.

In particular, in the enrichment of rare cells, for example circulating tumor cells with certain mutations, immune cells with defined epitopes and antigens or stem cells, lysis in a small volume is important.

In addition, the lysate should be able to be processed lossless. With the device 141, fluid can be drawn up into the cannula 148 with the aid of the rotary device 142, as can be seen in FIG. Possible lysate residues, for example individual drops, can remain in the cannula 148, as indicated in FIG. 16. As can be seen in FIG. 17, these lysate residues are displaced from the cannula 148 by the addition of oil until the complete lysate is collected in the container.

The feeding of the oil phase takes place via a fluid supply device 157, as indicated in FIG. 17 by an arrow 158. By a slow

Nachschieben the oil phase unwanted mixing in the two-phase system is prevented. Thus, the oil can then be decanted off by a phase separation or continue to be used as a seal.

If a certain amount of lysis buffer is placed in the container 156, only a part of this volume can be drawn in for lysis and, as described above, again completely into the preliminary storage vessel or the Container 156 are returned. Thus, a volume preservation is possible, whereby a complex Volumenadjustieren and consuming

Volume measurements can be omitted.

In FIGS. 18 and 19, it is shown that the oil phase is conveyed by means of a syringe 162 via the connection 153; 173 can be introduced into the device 141. The syringe 162 is advantageously designed as a Luer part and can instead of the closure body (155 in Figure 14) to the third port 153; 173 of the T-piece 150; 170 are bolted. Thus, syringe 162 may be considered

Fluid supply can be used. To apply the oil phase, a piston of the syringe 162 can be actuated. In FIG. 18, the syringe 162 is arranged orthogonal to the biopsy needle. In FIG. 19, the syringe 162 is arranged parallel to the biopsy needle due to the other embodiment of the T-piece 170.

FIGS. 20 to 23 illustrate a method in which the device 141 can advantageously be integrated into a lab-on-chip platform in a fluidic sequence for a lysis process with cells adhering to a biopsy needle. In the process schematically illustrated, a liquid phase 183 is pre-stored in a sample input chamber 182. The sample input chamber 182 is provided, for example, in a lab-on chip 181 of a microfluidic system 180.

The liquid phase or liquid 183 is a lysis buffer present in the volume to later realize a lyophilized bead of the chemicals for a subsequent analysis reaction, for example, sequencing. The biopsy needle device 141 described above (not shown in FIGS. 20 to 23) is then used to draw in so much lysis buffer 183 that the cannula is completely filled, as can be seen in FIG. The lysis can also by multiple high and

Pulling down lysis buffer are performed. The stroke when pulling up and down the lysis is initiated by rotation of the rotary body 147, as indicated in Figure 21 by an arrow 184.

Then, as indicated in FIG. 22 by an arrow 187, the lysate is completely expelled from the device 141 by oil 188. By phase separation is the now preserved lysis buffer volume including cell material in the sample input chamber 182, which is also referred to as an advance chamber. The lysis buffer volume is then additionally covered by the oil.

The sample input chamber 182 can, as indicated in FIG. 23 by arrows 191 and 192, be actuated by a suitable microfluidic system of the microfluidic system 180. In this case, a feeder channel 194 of the sample input chamber 182 is filled by means of oil 193. The lysate is then trapped between two oil phases and can be lost in the lab-on-chip system 181

be transported.

In Figures 24 to 26, the application is shown with a syringe 162 in which a first phase 201 and a second phase 202 is upstream. The first phase 201 is, for example, an aqueous phase. The second phase 202 is, for example, an oil phase. A

Phase separation is advantageously achieved by a parallel arrangement of the syringe 162, as shown in FIG. The desired phase separation ensures that the two phases 201, 202 in the syringe 162 do not mix.

In FIG. 24, a double arrow 204 indicates that the device 141 with the rotary device 142 can be used to draw in and expel fluid.

In FIG. 25, an arrow 211 indicates that first the aqueous phase 201, for example, is pushed through the cannula 148 and can be pumped up and down with the aid of the syringe 162, as indicated by arrows 212 to 214 in FIG. In this case, the lysis buffer and the lysate mix with the liquid 201. This is of particular interest when a lysis process is used, which consists of several steps. For example, basic lysis buffers are neutralized by an acid buffer before the lysate is further processed. In FIG. 26, an arrow 218 indicates that, to complete the process, the second phase or oil phase 202 is introduced from the syringe 162 into the device 141.

Claims

claims

Device (1; 41; 51; 71; 131; 141) for preparing sample material (33), characterized in that the device (1; 41; 51; 71; 131; 141) is designed as a rotating device (142) in that by means of a rotational movement a defined amount of liquid can be drawn into a sample receiving space (15) for the sample material (33) or expelled from the sample receiving space (15) for the sample material (33).

2. Apparatus according to claim 1, characterized in that the

Turning device (1; 41; 51; 71; 131; 142) comprises a rotary body (2; 42; 52; 72; 147), which via a thread (13; 43; 53; 73) with a

Liquid receiving space (5) is coupled to a volume whose size by rotating the rotary body (2; 42; 52; 72; 147) relative to the thread (13; 43; 53; 73) or by turning the thread (13; 53, 73) is changed relative to the rotary body (2; 42; 52; 72; 147).

3. Apparatus according to claim 2, characterized in that the

Liquid receiving space (5) by a hollow body (4; 44; 54; 74) is provided, which is internally provided with the thread (13; 43; 53; 73).

4. Apparatus according to claim 3, characterized in that the

Hollow body (4; 44; 54; 74) at a the sample receiving space (15) facing away from a sealed through-insertion region (38).

5. Apparatus according to claim 4, characterized in that the

sealed penetration area (38) has a sample carrying the needle (35).

6. Device according to one of claims 2 to 5, characterized in that the liquid receiving space (5) fluidly with the

Sample receiving space (15) communicates.

7. Device according to one of claims 2 to 6, characterized in that the sample receiving space (15) by a tubular body (10) is limited, which is open at its liquid receiving space (5) facing away from the end.

8. Apparatus according to claim 7, characterized in that the

Tubular body (10) or the rotary body (2; 42; 52; 72) one

Having externally threaded portion (7) which is complementary to a

Inside threaded portion (6) is formed in the

Liquid receiving space (5) is arranged.

9. Apparatus according to claim 7 or 8, characterized in that the tubular body (10), the hollow body (4; 44; 54; 74) and / or the rotary body (2; 42; 52; 72) are provided with at least one sealing device (45 ; 55; 75,76; 132) are / is combined.

10. Apparatus according to claim 7 or 8, characterized in that the tubular body (10), the hollow body (4; 44; 54; 74) and / or the rotary body (2; 42; 52; 72) with a filter device (32; 120) are combined.

11. Device according to one of claims 2 to 10, characterized in that the liquid receiving space (5) or the sample receiving space (15) has a connection (40; 153; 173) for supplying and / or discharging a fluid.

12. The device according to claim 11, characterized in that the

A port (40; 153; 172) for supplying and / or discharging the fluid is provided as a third port on a T-piece (150; 170) delimiting the fluid receiving space (5) and / or the sample receiving space (15).

The apparatus according to claim 12, characterized in that the tee (150; 170) has a first port (151; 171) for the rotating device, a second port (152; 172) for the sample receiving space (15) and the third port (153; 173) for supplying and / or discharging the fluid.

14. A microfluidic system (100) comprising means (1; 41; 51; 71; 131; 141) for preparing sample material (33) according to any one of the preceding claims.

15. A method of preparing sample material with a device

(1; 41; 51; 71; 131; 141) according to one of Claims 1 to 13.