WO2021013595A1

WO2021013595A1 - Lab-on-a-chip system comprising at least one functionalized section

Info

Publication number: WO2021013595A1
Application number: PCT/EP2020/069692
Authority: WO
Inventors: Hannah Bott
Original assignee: Robert Bosch Gmbh
Priority date: 2019-07-19
Filing date: 2020-07-13
Publication date: 2021-01-28
Also published as: DE102019210697A1

Abstract

The invention relates to a lab-on-a-chip system (1) comprising at least one functionalized section (14, 41, 51) which is designed for the adhesion of sample components (32), wherein the functionalized section (14, 41, 51) has a defined length and/or a defined surface area in order to facilitate a quantifiable isolation of sample components (32).

Description

description

title

Lab-on-chip system with at least one functionalized section

The invention relates to a lab-on-chip system with at least one

functionalized section and a method for using the lab-on-chip system. In particular, a microfluidic system and a microfluidic process for the quantitative on-chip isolation of sample material are specified.

State of the art

Microfluidic systems allow the analysis of small amounts of samples with a high level of sensitivity. The automation, miniaturization and

Parallelization of the processes also allows a reduction in manual steps and a reduction in errors caused by them.

This makes it possible to examine and evaluate a patient sample, such as blood, directly at the point of care. A fast sample analysis should be aimed for, without complicated and time-consuming work steps that would have to be carried out by trained personnel in central laboratories. This can be implemented using a lab-on-chip (LoC) system. LoC systems are used for various applications. In a network system of channels and chambers on a microfluidic basis, the various problems can be processed and different processes can be programmed. When designing a LoC system, it is desirable, among other things, to enable the analytes to be isolated from the sample that is placed in the input chamber of the chip, the so-called world-to-chip interface. The samples can be complex

Multicomponent fluids such as blood, urine, breath condensate or liquor. For example, blood is the carrier material of the sample to be analyzed in many in-vitro diagnostic tests. In order to isolate the appropriate sample material from the blood for analysis, several manual purification steps are usually carried out in conventional laboratory operations. This includes the lysis of cells in the starting material, the connection of the sample material, such as DNA, to a filter, as well as the washing and elution of the isolated sample material. In particular, the amount of sample present is usually also determined during the purification by measuring the number of cells. This must be carried out in particular if a defined amount of sample is required for the subsequent detection reaction in order to be able to make a quantitative statement. In addition, it is also possible to bring the concentration of the sample into the range of a predetermined starting concentration by dilution.

It is therefore a requirement of a LoC system to integrate the steps for purifying a sample into the microfluidic network. However, this often requires a redesign of the microfluidic components. A universal platform for various applications is therefore associated with high development costs. Possible solutions to circumvent this problem are a partial surface functionalization of the system, an adapted one

Fluid guidance and the use of different fluid phases. This enables multi-stage, dynamic processes, for example for the isolation of

Sample material, can be integrated on a LoC system.

Disclosure of the invention

A LoC system is proposed here with at least one functionalized section that is used for the adhesion of sample components (in particular

Sample molecules), wherein the functionalized section has a defined length and / or a defined area to a

quantifiable isolation of sample components (in particular

Sample molecules).

The LoC system is usually a microfluidic system, in particular for receiving and / or processing a (patient) sample. Corresponding microfluidic systems are used especially for handling and / or processing a sample (directly) at the so-called “point-of-care”.

The at least one functionalized section has a (pre-) defined length and a (pre-) defined area. The functionalized section can preferably be covered with a layer of surface molecules or

Adhesion molecules be provided. The surface molecules or

Adhesion molecules are usually provided and set up (specifically) to hold the sample molecules to be isolated in the section.

The sample components are in particular

Sample components of a certain type or variety that are to be specifically isolated from the sample. The sample components can be sample molecules (of a certain type or variety), for example. As a rule, the sample components are sample molecules that are to be specifically isolated from the sample. Alternatively or additionally, the

Sample components are cells.

In particular if a defined or known density of surface molecules or adhesion molecules is contained in the layer, in the case of a usually also defined or known layer thickness (and / or defined or known layer volume) from the defined length or area of the functionalized section (directly) on the (largely exact) number of surface molecules or adhesion molecules in the section

be inferred. In other words, the functionalized section with its defined length / area is used in particular to determine the number of cells (in particular the maximum number of cells that can be isolated and / or to be isolated).

This is automated and simultaneously in an advantageous manner

enables quantifiable purification of a sample in a LoC system. The sample can also be transferred to another medium of known volume. The steps mentioned can either already be implemented in the world-to-chip interface, i.e. in the sample input chamber, or in the adjoining part of the microfluidic system.

A particularly advantageous aspect of the LoC system specified here is the defined surface coating of a specified area of the LoC system. System, and / or the use of different fluid phases and a suitable fluid flow of these phases. The surface coating can be formed with adhesion molecules that can specifically bind the sample molecules to be separated. In addition to the sample liquid, inert fluids can be used as the fluid phases as washing buffers and / or carrier reagents which detach the attached sample molecules or, in the case of cells, can lyse them. A suitable fluid flow is generally dependent on the diffusion coefficient of the to be separated

Sample molecules and can therefore be adjusted in such a way that enough particles can diffuse to the functionalized surface and be bound there so that it can be saturated with sample molecules.

One advantage of the approach described here of a functionalized section of defined length and / or area (to determine the number of cells) is that it can be used universally in many microfluidic systems.

In particular if there is a functionalizable channel section through which the sample and various reagents can be pumped.

The described approach of a functionalized section of defined length and / or area can be used on an already existing LoC platform. The range of functions can thus be expanded without having to adapt the core of the fluidics or the manufacturing process of the chip.

One or more (different from one another) surface coatings can be provided in the at least one functionalized section. In particular through the use of different

Surface coatings that specifically bind different analytes, several samples can be isolated and processed simultaneously on the same chip.

Furthermore, it is conceivable that the channel surfaces of the entire fluidic system can be functionalized at certain points and / or used for processing the fluids. Areas that should remain untouched for later analysis can be left out. In addition, a sequential processing of the upstream reagents, fluids and the lyophilizate can advantageously be implemented on the LoC. This can enable the required components to be added at the appropriate time.

With the approach proposed here, the risk of the disintegration of fragile sample material can furthermore advantageously be reduced. This is in particular because steps for sample preparation can be integrated on the LoC and thus the time-consuming, manual off-chip steps can be omitted.

Furthermore, the isolation of the sample material can advantageously be standardized for many samples. Different steps can be on-chip or in the

Sample entry chamber can be performed. This can do for many

various LoC applications can be advantageously achieved that no additional training of the staff is necessary.

In particular, the functionalization of a defined channel area in the case of a saturated connection of sample material enables the isolated sample to be quantified. This is a considerable advantage, especially for quantitative analyzes, since no complex counting steps have to be implemented on the chip.

It can also be provided that the carrier reagent with which the analytes are released from the functionalized channel section and transported to the reaction chamber has a significantly smaller volume than the original volume of the sample to be analyzed. Thus, not only can the analytes be isolated, but also advantageously a volume reduction, which can be advantageous for many microfluidic analyzes.

Furthermore, in particular, a dilution of the sample material is also possible, in particular if a larger volume of carrier reagent is used. In addition, no filter is advantageously required on the chip for the approach presented, its installation in terms of production technology and its

Controlling fluidically can be a challenge.

According to an advantageous embodiment, it is proposed that the

functionalized section is arranged in a flow channel. The The flow channel generally describes a (microfluidic) channel through which the sample can flow.

In this context, it is provided in particular that the

Flow channel forms a fluidic connection between at least one input reservoir and at least one output reservoir of the LoC system. The input reservoir can be, for example, an input chamber through which the sample can be introduced or input into the (microfluidic) LoC system. In which

The output reservoir can be an outlet or a

Act processing chamber of the LoC system.

In this context, a cross section of the

Flow channel be at least a factor of ten (10) smaller than a cross section of the at least one input reservoir and / or the at least one output reservoir.

In particular, the LoC system can have at least one first input reservoir and one second input reservoir and at least one first output reservoir and at least one second output reservoir. In addition, the LoC system can also have at least one valve arrangement with which the input reservoirs and / or the output reservoirs can each be specifically connected to the flow channel.

It is preferred that the functionalized section is a functionalized one

Has surface with adhesive surface molecules. For this purpose, the functionalized surface can be coated with adhesion molecules, for example, so that analytes can adhere there. For this purpose, for example

Fibronectin or poly-D-lysine can be used.

Furthermore, it can be provided that at least one first section with a first functionalized surface with a first function and at least one second section with a second functionalized surface with a second function are arranged along the flow channel in a flow direction, the first function and the second Differentiate between functions. In this context, it can also be provided that the

Flow channel is connected to an intermediate reservoir between the first section and the second section. The intermediate reservoir can (optionally) be an input reservoir and / or a

Trade output reservoir.

According to a further advantageous embodiment, it is proposed that the functionalized section be formed in an input chamber of the LoC system. If several functionalized sections are provided, it can be provided in this context that at least one of these sections is formed in the input chamber.

According to a further aspect, a method for the quantifiable isolation of sample constituents with a LoC system described here is also proposed, a flow rate of a sample being selected in such a way that adherence of sample constituents in the functionalized section is promoted. To facilitate this, the flow rate can be adjusted, for example, so that a minimum number of sample components can adhere.

In this context, it is also advantageous if the flow rate is chosen such that the functionalized section is saturated with sample constituents. Furthermore, it can be provided that a diffusion coefficient of sample components in the sample is used when determining the

Flow velocity is taken into account. In addition, it can also be provided that a radius of the sample components when determining the

Flow velocity is taken into account.

As an alternative or in addition, at least one of the following parameters can be taken into account when determining the flow rate or set specifically for the adherence of sample components in the functionalized section:

Viscosity of the sample,

Temperature of the sample.

The details, features and advantageous configurations discussed in connection with the LoC system can also be used here presented procedures occur and vice versa. In this respect, reference is made in full to the statements made there for a more detailed characterization of the features.

The solution presented here and its technical environment will be

explained in more detail below with reference to the figures. It should be pointed out that the invention is not intended to be restricted by the exemplary embodiments shown. In particular, unless explicitly stated otherwise, it is also possible to extract partial aspects of the facts explained in the figures and to combine them with other components and / or findings from other figures and / or the present description. It shows schematically:

1: a first example of a lab-on-chip system described here,

FIG. 2: a possible microfluidic sequence of a sample purification with the system from FIG. 1,

Fig. 3: a detailed view of a flow channel for one here

described system,

4: a second example of a lab-on-chip system described here, and

FIG. 5: a third example of a lab-on-chip system described here with an associated, exemplary microfluidic process.

1 schematically shows a first example of a lab-on-chip system 1 described here. The lab-on-chip system 1 has a functionalized section 14, which is set up for the adhesion of sample constituents 32, the functionalized section 14 has a defined length and / or a defined area in order to achieve a quantifiable isolation of

To enable sample components 32.

The functionalized section 14 is exemplary in one

Flow channel 2 arranged. In addition, the throughflow channel 2 forms, for example, a fluidic connection between two

Input reservoirs 10, 17 and two output reservoirs 15, 16 of the lab-on-chip system 1. It is also indicated in Fig. 1 that a cross section of the

Flow channel 2 is at least a factor of 10 smaller than a cross section of the at least one input reservoir 10, 17 and / or the at least one output reservoir 15, 16.

The lab-on-chip system 1 according to FIG. 1 has, for example, a first

Input reservoir 10 and a second input reservoir 17 and a first output reservoir 15 and a second output reservoir 16. In addition, the lab-on-chip system 1 has a valve arrangement 11 with which the

The input reservoirs 10, 17 and / or the output reservoirs 15, 16 can each be specifically connected to the throughflow channel 2.

In other words, FIG. 1 shows, in particular, a basic example of the presented lab-on-chip system 1. The system 1 has a reservoir for sample input 10, a pump unit 11 and at least one

Reaction chamber 15, a waste chamber 16 and a pre-storage chamber 17. The inlet to the chambers can be closed by a valve 12, 13. In addition, a defined part 14 of the microfluidic channel system is functionalized, that is, the channel surface has been coated with adhesion molecules so that analytes can adhere there. For example, fibronectin or poly-D-lysine can be used for this purpose. The functionalization of the channel section is carried out, for example, in such a way that the area and the concentration of the adhesion molecules on the surface are matched to the requirements of the application and are known.

2 shows schematically a possible microfluidic sequence of a

Sample purification with the system from FIG. 1. For this purpose, FIG. 2 is subdivided into FIGS. 2A to 2E, which describe an exemplary sequence.

In FIG. 2A, a sample 20 is placed in the input reservoir 10. In the

Pre-storage chamber 17 is given a carrier medium.

In FIG. 2B, the sample 20 is pumped through the functionalized channel region 14 into the waste chamber 16. Here, the flow rate is set, for example, so that the functionalized surface is completely saturated with sample molecules. The determining factor for the maximum flow velocity is in particular the diffusion coefficient of the sample to be separated, which in addition to the hydrodynamic radius of the sample molecules is also dependent on the temperature and the dynamic viscosity of the sample liquid. If these parameters are known, the maximum

Flow rate can be determined (e.g. modeled), otherwise it can be determined experimentally by determining the purification efficiencies at different flow rates.

Thus, FIG. 2 also illustrates a method for the quantifiable isolation of sample components 32 with the lab-on-chip system, in which a

The flow rate of a sample 20 is selected in such a way that adhesion of sample constituents 32 in the functionalized section is promoted. As described, the flow rate for this can be selected in such a way that the functionalized section 14 is saturated with sample constituents 32. A diffusion coefficient of sample components 32 in sample 20 can be taken into account when determining the flow rate. In addition, a radius of the sample components 32 can be used when determining the

Flow velocity is taken into account. Furthermore, at least one of the following parameters can also be taken into account when determining the flow rate or can be set specifically for the adherence of sample constituents 32 in the functionalized section 14, 41, 51:

Viscosity of the sample,

Temperature of the sample.

In Fig. 2C, the input reservoir 10 is emptied. Some of the analytes from the sample material are bound to the specifically adhesive surface of the functionalized channel region 14, this being saturated, that is to say all adhesive binding sites are occupied with sample molecules. The remaining sample material is located in the waste chamber 16. If necessary, the channel can also be rinsed at this point with a washing buffer, which removes unattached sample molecules and transports them into the waste chamber.

In FIG. 2D, the carrier reagent 21 is transferred from the storage chamber 17 via the functionalized channel region 14 into the reaction chamber for the

Detection reaction 15 is pumped. Depending on the target and the principle of adhesion, this can be a lysis buffer or a solvent that detaches the analytes again and transports them into the reaction chamber. In FIG. 2E, the carrier reagent 21 is completely pumped out of the storage chamber through the functionalized channel region 14 into the reaction chamber 15. This sets a defined sample volume. Due to the previously ensured saturation of the binding of analytes to the surface of the functionalized channel area, the concentration of sample material in the carrier reagent is now known and a quantifiable one can be used

Detection reaction can be carried out.

3 schematically shows a detailed view of a flow channel 2 for a system described here. It is exemplified in FIG. 3 that the functionalized section 14, 41 can have a functionalized surface with adhesive surface molecules 31, 51. An exemplary sequence is also shown in FIGS. 3A to 3E in FIG.

In this context, FIG. 3 shows the corresponding detailed views of the functionalized channel region 14 from FIGS. 2A to 2E. In this

For example, cells are isolated from the starting material and lysed in order to carry out a genetic analysis of the DNA.

3A shows a detailed view 14 of the functionalized channel section 30. In this region of the microfluidic channel, the inner surface of the channel is functionalized with adhesive surface molecules 31.

In FIG. 3B, the sample 20 is pumped from the input chamber through the microfluidic channel with the functionalized section 14 into the waste chamber. The cells 32 contained in the sample are to be isolated for genetic analysis and are attached to the surface by the adhesive molecules

Channel surface bound.

In Fig. 3C it is shown that with a sufficient concentration of analytes in the sample, the surface of the functionalized channel section after the

Pumping through the sample liquid is saturated with analytes. In this case, for example, a quantifiable analysis is possible because the number of cells is known. In FIG. 3D, the carrier reagent 21 for the target connected in the functionalized channel section 14 is then pumped from the pre-storage chamber into the reaction chamber. In this case, the carrier reagent contains a lysis medium, so that the bound cells 33 are lysed when rinsing through and the DNA strands 34 get into the carrier medium. At a

When the functionalized surface is saturated with cells, the amount of DNA and thus also the concentration in the carrier reagent is known. Will have a significantly lower volume in relation to the original sample material

When the carrier reagent is used, in addition to the isolation and quantification of the analyte, there is also a volume reduction, which for many microfluidic

Analysis can be beneficial.

In Fig. 3E, the lysed cells 35 remain on the channel wall. The

Carrier reagent with DNA is located in the reaction chamber and can be analyzed.

4 schematically shows a second example of a lab-on-chip system 1 described here. In this case, at least one first section 14 with a first functionalized surface with a first function and at least one second section are exemplarily along the flow channel 2 in a flow direction 41 with a second functionalized surface with a second function, wherein the first function and the second function differ from one another.

Furthermore, it is exemplified in FIG. 4 that the

Flow channel 2 can be connected to an intermediate reservoir 15 between the first section 14 and the second section 41.

FIG. 4 particularly illustrates the scalability of the concept for samples which contain several targets to be analyzed. There are several

Channel sections one after the other with adhesive that is selective for the target

Functionalized surface molecules. Between the functionalized

The reaction chambers for the detection reaction of the respective target are located in channel sections.

An exemplary fluidic sequence for the parallel processing and analysis of several targets from a sample can be broken down as follows: (A) The sample from the input chamber 10 is pumped through the microfluidic channel with the functionalized sections 14, 41,... Into the waste chamber 16. The valves to the reaction chambers 12 are closed, the valves along the channel to the waste chamber 13 are open.

(B) In the following, the carrier reagent for the target connected in the first channel section 14 is pumped from the first pre-storage chamber 17 into the first reaction chamber 15.

(C) In the next step, the carrier reagent for the target connected in the second channel section 41 is pumped from the second pre-storage chamber 40 into the second reaction chamber 42.

(D) This step can be repeated x times for further targets, a storage chamber, a functionalized channel section and a reaction chamber are added.

(E) The samples can now be analyzed and the detection reactions can take place in the respective reaction chambers.

5 schematically shows a third example of a lab-on-chip system described here with an associated, exemplary microfluidic process. The functionalized section 51 is formed, for example, in an input chamber 10 of the lab-on-chip system 1.

In this context, FIG. 5 shows, in particular, how the described microfluidic system and method also directly in one

Sample input chamber (a lab-on-chip system) can be implemented. For this purpose, a microfluidic polychamber, for example, can be used as a world-to-chip or sample input chamber.

Thanks to the microfluidic control of the polychamber and the

The following, exemplary sequence according to FIGS. 5A to 5G for isolating the sample material can be made possible: In Fig. 5A, a sample entry adapter 50 having two chambers is employed. A carrier medium 21 is pre-stored in the pre-storage chamber 17, and in the input chamber 10 there is an area 51 which is filled with adhesives

Surface molecules is functionalized.

In FIG. 5B, the sample 20 is placed in the input reservoir 10.

In FIG. 5C, the sample 20 is drawn into the downstream part of the microfluidic system, e.g. B. via a peristaltic pumping mechanism. A portion 33 of the analytes from the sample is used by the adhesive

Surface molecules specifically bound.

In FIG. 5D the input chamber 10 is emptied, a part 33 of the analytes from the sample material is bound to the specifically adhesive surface of the functionalized area 51. The remaining sample material is located in a waste chamber of the microfluidic system 1.

In FIG. 5E, the carrier reagent 21 is now removed from the preliminary storage chamber 17 by an underlayer principle with a non-miscible phase 52, e.g. B. silicone oil, transferred into the input chamber 10. In this way it can be achieved that an exact volume of carrier reagent is transferred into the input chamber. In particular, this allows the amount and concentration of analytes to be set exactly if the amount of analytes is known when the binding of analytes to the surface of the functionalized area is saturated.

In FIG. 5F, the carrier reagent 21 for the target connected in the functionalized area 51 is in the input chamber. In this case, the carrier reagent contains a lysis medium, so that the bound cells 33 are lysed and the DNA strands 34 get into the carrier medium. When the functionalized surface is saturated with cells, the amount of DNA and thus also the concentration in the carrier reagent is known. Will have a significantly lower volume in relation to the original sample material

Analysis can be beneficial. In Figure 5G, the lysed cells 35 remain on the functionalized area in the input chamber. The carrier reagent with DNA is drawn into the fluidic system and can be analyzed.

Material examples for the microfluidic system are, in particular, polymers such as polycarbonate (PC), polypropylene (PP), polyethylene (PE), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS) or

thermoplastic elastomers (TPE) such as polyurethane (TPU) or styrene block copolymer (TPS), in particular manufactured using high-throughput processes such as injection molding, thermoforming, stamping, laser transmission welding.

The microfluidic pump device and valves can be implemented, for example, by the pneumatically actuated deflection of a polymer membrane in recesses in a polymer substrate in which the

microfluidic channels and reaction chambers can be located.

An exemplary dimensioning of the microfluidic system can be given as follows:

- Thickness of the polymer substrates: 1 mm - 10 mm

- Thickness of the polymer membrane: 100 pm - 300 pm

- Duct cross-sections: 100 x 100 pm ² - lxl mm ²

- Chamber volumes: 1 pl - 100 pl

Possible sample liquids are in particular aqueous solutions,

especially for carrying out chemical, biochemical, medical or molecular diagnostic analyzes, e.g. B. body fluids, smears, secretions, sputum or tissue samples.

Preferred targets are, in particular, sample material contained in the sample liquids, in particular of human origin, e.g. B. bacteria, viruses, certain cells, such as. B. circulating tumor cells, cell-free DNA, proteins or other biomarkers.

Claims

Expectations

1. Lab-on-chip system (1) with at least one functionalized

Section (14, 41, 51) which is set up for adhesion of sample components (32, 33), the functionalized section (14, 41, 51) having a defined length and / or a defined area in order to achieve quantifiable isolation of sample components (32, 33) to enable.

2. lab-on-chip system (1) according to claim 1, the functionalized

Section (14, 41) is arranged in a flow channel (2).

3. lab-on-chip system (1) according to claim 2, wherein the

Through-flow channel (2) a fluidic connection between at least one input reservoir (10, 17) and at least one

Forms output reservoir (15, 16) of the lab-on-chip system (1).

4. lab-on-chip system (1) according to claim 2 or 3, wherein a cross section of the flow channel (2) is at least a factor of 10 smaller than a cross section of the at least one input reservoir (10, 17) and / or the at least a dispensing reservoir (15, 16).

5. lab-on-chip system (1) according to one of claims 2 to 4, wherein the lab-on-chip system (1) has at least a first input reservoir (10) and a second input reservoir (17) and at least a first one

Dispensing reservoir (15) and at least one second dispensing reservoir (16), as well as at least one valve arrangement (11), with which the input reservoirs (10, 17) and / or the output reservoirs (15, 16) are each targeted to the flow channel (2) can be connected.

6. lab-on-chip system (1) according to one of claims 2 to 5, wherein the functionalized section (14, 41) has a functionalized surface with adhesive surface molecules (31, 51).

7. lab-on-chip system (1) according to any one of claims 2 to 6, wherein along the flow channel (2) in a flow direction

at least a first section (14) with a first functionalized Surface with a first function and at least one second

Section (41) with a second functionalized surface with a second function are arranged, wherein the first function and the second function differ from one another.

8. lab-on-chip system (1) according to claim 7, wherein the

Flow channel (2) between the first section (14) and the second section (41) is connected to an intermediate reservoir (15).

9. lab-on-chip system (1) according to one of the preceding sections, wherein the functionalized section (51) is formed in an input chamber (10) of the lab-on-chip system (1).

10. A method for the quantifiable isolation of sample components (32) with a lab-on-chip system (1) according to one of the preceding

Claims, wherein a flow rate of a sample (20) is selected in such a way that adherence of sample components (32) in the functionalized section (14, 41, 51) is promoted.

11. The method of claim 10, wherein the flow rate is such

it is chosen that the functionalized section (14, 41, 51) with

Sample components (32) is saturated.

12. The method according to claim 10 or 11, wherein a diffusion coefficient of sample components (32) in the sample (20) is taken into account when determining the flow rate.

13. The method according to any one of claims 10 to 12, wherein a radius of the sample components (32) is taken into account when determining the flow rate.

14. The method according to any one of claims 10 to 13, wherein at least one of the following parameters is taken into account when determining the flow rate or is specifically set for the adhesion of sample components (32) in the functionalized section (14, 41, 51):

Viscosity of the sample,

Temperature of the sample.