WO2020002396A1

WO2020002396A1 - Microfluidic apparatus and method for verifying a target protein of a biological sample

Info

Publication number: WO2020002396A1
Application number: PCT/EP2019/066948
Authority: WO
Inventors: Katrin Luckert
Original assignee: Robert Bosch Gmbh
Priority date: 2018-06-29
Filing date: 2019-06-26
Publication date: 2020-01-02
Also published as: DE102018210710A1

Abstract

The invention relates to an apparatus (100), in particular to a microfluidic apparatus (100), and a method (500) for verifying a target protein (10) of a biological sample, comprising a sample chamber (111) for receiving the biological sample, a first antibody (121) and a second antibody (122), wherein the first antibody (121) and the second antibody (122) are designed to bind to the target protein (10) at different locations and wherein the first antibody (121) has a specified first nucleic acid fragment (151) and the second antibody (122) has a specified second nucleic acid fragment (152), furthermore comprising a verification chamber (105), fluidically connected to the sample chamber (111), for verification of a third nucleic acid fragment (153) as verification of the target protein (10), wherein the third nucleic acid fragment (153) is formed in the sample during a binding of the first nucleic acid fragment (151) to the second nucleic acid fragment (152) during binding of the first antibody (121) and of the second antibody (122) to the target protein (10), with the addition of ligase (128) or polymerase (128) and in the presence of unbound nucleotides (125).

Description

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description

title

Microfluidic device and method for the detection of a target protein of a biological sample

State of the art

The breath contains over 3000 different components of different chemical nature. In addition to gases and moisture, the breath contains non-gaseous aerosols as carriers for non-gaseous components such as

Proteins, metabolites and nucleic acids, which are considered disease-specific

Biomarkers allow conclusions to be drawn about an illness or a therapeutic success. According to the state of the art, the aerosols or their ingredients come from the lung-lining liquid film, so that one

Analysis of the collected aerosols consequently opened a non-invasive analysis method.

These biomarkers, in particular the proteins, can be detected by analyzing the breathing gas condensate. For example, in Gullberg and Andersson, Nature Methods 7, (2010) doi: 10.1038 / nmethJ.306

sensitive protein detection described in the form of a protein ligation assay.

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Disclosure of the Invention Advantages of the Invention

Against this background, the invention relates to a device for the detection of a target protein in a biological sample. The device comprises one

Sample chamber for receiving the biological sample and a first one

Antibody and a second antibody. The first antibody and the second antibody are designed to be different from the target protein

Tie bodies. Furthermore, the first antibody has a predetermined first nucleic acid fragment and the second antibody has a predetermined second

Nucleic acid fragment. The device also includes one with the

Sample chamber fluidly connected detection chamber for detection of a third nucleic acid fragment as detection of the target protein, the third nucleic acid fragment when the first nucleic acid fragment binds to the second nucleic acid fragment when the first antibody and the second antibody bind to the target protein with addition of a ligase or a polymerase and under Presence of unbound nucleotides is formed in the sample.

The device is to be understood in particular as a microfluidic device, also called a lab-on-a-chip. The target protein can be a protein of a specified type or of a specified type or of a specified type, in particular a protein whose occurrence in the sample indicates an infection or disease of the sampler. The first antibody and the second antibody can in particular be two different types, types or types of antibodies.

In particular, the first antibody has a first binding site and the second antibody has a second binding site for binding to the target protein, the first binding site being different from the second binding site

differs so that the first antibody and the second antibody

bind to the target protein at different locations. In particular, the first binding site is part of the first nucleic acid fragment and the second

Binding site part of the second nucleic acid fragment. The device comprises R. 371836

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- 3 - preferably a plurality of the first antibodies and a plurality of the second antibodies. A first antibody or a second antibody is therefore to be understood in particular as a multiplicity of first antibodies or second antibodies. Under a

Nucleic acid fragment is in particular part of a nucleic acid,

for example part of a DNA or RNA. Under the first

Nucleic acid fragment and the second nucleic acid fragment

to understand in particular two different nucleic acid fragments. With the addition of a ligase or a polymerase is in particular the addition

to understand several ligase enzymes or polymerase enzymes.

The invention advantageously enables protein detection, in particular in the form of a protein ligation assay or a protein extension assay, in a particularly microfluidic device

well defined and reliable. Furthermore, the

Device according to the invention advantageously an uncomplicated

Detection of the third nucleic acid fragments formed in the detection chamber. For this purpose, the detection chamber can, for example, be a microarray for a

Have hybridization of the third nucleic acid fragments to the microarray. Alternatively, an optical readout via an optical interface of the

Detection chamber, especially with the help of a

Fluorescence spectroscopy. The invention thus advantageously enables parallel, in particular multiplex, detection of several target proteins.

The device, in particular the detection chamber or the first chamber, is preferably set up to carry out a polymerase chain reaction (PCR for short) of the third nucleic acid fragments and, for this purpose, comprises, for example, the aforementioned microarray for detecting the third nucleic acid fragments reproduced via the PCR. This has the advantage that even very low concentrations of target proteins can be effectively detected in the sample, which is particularly important for proteins in a sample

Breathing gas condensate may be the case. The use of such a PCR-based readout device has the advantage of signal amplification, that is to say few binding processes of antibodies and target protein can be multiplied by multiplication of a nucleotide sequence (PCR) and thus leave R. 371836

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- 4 - advantageously a very sensitive detection reaction. Alternatively, the device, in particular the detection chamber or the first chamber, is set up to carry out a quantitative real-time PCR (abbreviated to qPCR) and, for this purpose, comprises the optical interface described above for optical detection of the third ones reproduced via the real-time PCR

Real time nucleic acid fragments.

According to an advantageous development of the invention, the first

Nucleic acid fragment on a first nucleotide sequence which is complementary to a second nucleotide sequence of the second nucleic acid fragment.

In particular, the first nucleotide sequence is the first

Nucleic acid fragment and the second nucleotide sequence of the second

Nucleic acid fragments freely accessible, preferably by the

Nucleotide sequences are arranged at one end of the first and the second nucleic acid fragment. This enables a protein extension assay with the device according to the invention in a simple manner

perform.

According to a further advantageous development of the invention, the first nucleotide sequence and the second nucleotide sequence are complementary to two further predetermined nucleotide sequences at two locations each. This has the advantage that a protein ligation assay can be carried out with the device, the further nucleotide sequences in unbound form to the antibodies and the target protein for a circular PCR amplification

can be added.

The first antibody is preferably immobilized in the sample chamber or in a first chamber of the device. This means in particular that the first antibodies on a solid phase of the sample chamber

or the first chamber are bound. The solid phase can in particular be a surface, for example a surface of a

Soil, the chamber or a microsphere such as particles or beads present in the chamber, which are usually used for immobilization in microfluidic systems, for example Dynabeads®. The particles or beads are preferably R. 371836

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- 5 - Magnetic or magnetizable particles or beads, so that they can be easily held in a desired chamber with a magnet. By immobilizing the first antibodies, the target proteins can advantageously be bound to the first

Antibodies are also immobilized in the first chamber and thus on

well-defined position. This particularly facilitates one or more rinses or washing steps to remove unwanted ones

Components. The immobilization can preferably be carried out via a

covalent binding of the first antibodies, in particular via a peptide bond formed by COOH groups of the solid phase with amino groups of the

Antibodies can be realized. Alternatively, an indirect binding of the first

Antibodies to the solid phase via a protein take the form of a protein-protein bond, for example via the protein A known from the cell wall of the bacterium Staphylococcus aureus. The protein can also be an antibody, for example a species

specific antibodies, for example isolated from a mammal such as the mouse.

As stated, the first antibody can also be immobilized in the sample chamber or the detection chamber instead of in the first chamber, in particular for immobilizing the target protein at the location of the detection. In other words, the first chamber with the sample chamber is in this case

or the detection chamber are identical, so that the protein detection takes place in the sample chamber or in the detection chamber.

Alternatively, the first chamber is fluidly connected to the sample chamber in order to convey the target proteins incorporated into the device together with the sample into the first chamber. In this case, the first chamber can be fluidly connected via the sample chamber or directly to the detection chamber.

The second is in a particularly advantageous development of the invention

Antibodies upstream in a second chamber of the device, for example in a storage chamber set up for this purpose. The storage chamber and / or the other chambers in which the antibodies can be stored can have a biocompatible coating for reliable storage. The antibodies can be lyophilized as a solid or in solution R. 371836

WO 2020/002396 PCT / EP2019 / 066948 6 can be stored, with the antibody being able to be detached by means of microfluidic control when stored in a dry place. The antibodies are preferably stored together with physiological buffer salts for stable storage. This development has the advantage that the second antibodies can be stored separately from the first antibodies and in particular can only be added after the target proteins have bound to the first antibodies.

This prevents premature formation of the third nucleic acid fragments.

In a particularly preferred development of the invention, the

Device the unbound nucleotides and preferably primers and

Enzymes for the formation of the third nucleic acid fragments. These can preferably also be upstream in one chamber, in particular in the second chamber or in a third chamber.

According to a particularly advantageous development of the invention, the device comprises reaction components for the enzyme functions of the ligase and / or the polymerase. These reaction components are in particular physiological buffers and co-factors as well as free nucleotides. The reaction components can preferably also in one

Chamber, in particular in the second or third chamber or in a fourth chamber. The ligase and / or polymerase can

for example in the second chamber or separately in a fifth chamber.

The invention also relates to a method for the detection of a

Target protein of a biological sample with a device, in particular with a microfluidic device. In a first step of the method, the biological sample is taken up in a sample chamber of the device. In a second step of the method, the sample is contacted with a first antibody and with a second antibody, the first

Antibodies and the second antibody are designed to bind to the target protein at different locations and the first antibody

have a predetermined first nucleic acid fragment and the second antibody has a predetermined second nucleic acid fragment. In a third step, a ligase or a polymerase is added which, when the R. 371836

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- 7 - first antibody and the second antibody to the target protein and in the presence of unbound nucleotides to form a third

Nucleic acid fragment causes a binding of the first nucleic acid fragment with the second nucleic acid fragment. In a fourth step, a

Detection of the third nucleic acid fragment formed as detection of the

Target protein in a detection chamber of the device, preferably after a polymerase chain reaction for duplication of the third

Nucleic acid fragment.

The advantages of the method according to the invention are set out in the above

corresponding advantages of the device according to the invention.

Brief description of the drawings

Exemplary embodiments of the invention are shown schematically in the drawings and are explained in more detail in the description below. The same reference numerals are used for the elements shown in the various figures and acting in a similar manner, and the elements are not repeated.

Show it

FIG. 1 shows an exemplary embodiment of the device according to the invention,

Figure 2a and b is a schematic representation of the protein extension Assaya

or protein ligation assay in the context of

Invention and

Figure 3 shows an embodiment of the method according to the invention.

Embodiments of the invention

FIG. 1 shows an exemplary embodiment of the device 100 according to the invention in the form of a microfluidic device 100 for detecting one or more R. 371836

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Target proteins. The device 100 has a first opening 110

accessible sample chamber 111, which is fluidly connected to a first chamber 101. The first chamber 101 comprises first antibodies 121, which are immobilized on a solid phase 131, for example beads 131.

The device 100 preferably comprises a magnet 140 which is arranged near the first chamber 101 for movement or fixation of the beads 131. The binding of the first antibodies 121 to the solid phase 131 can preferably be achieved by covalent binding of the first antibodies, in particular via COOH groups. Alternatively, the first antibodies 121 can be indirectly bound to the solid phase 131 via a protein 132, for example a protein-protein binding. The protein 132 can also be an antibody, for example a species-specific antibody.

The first chamber 101 is fluid with a second chamber 102

Device 100 connected, wherein in the second chamber 102 second antibodies 122 are upstream. The second chamber 102 further comprises primers 127, in particular for carrying out a protein ligation assay or a protein extension assay. A third chamber 103 of the device 100 is fluidly connected to the second chamber 102 and, in addition to a ligase 128 or polymerase 128, can comprise nucleotides 125 and / or reaction components 129 for the enzyme functions of a ligase and / or a polymerase, in particular buffers and co- factors. Alternatively, the

Reaction components can also be stored separately in separate chambers 107, 108, 109, in particular to enable stable storage and to prevent premature mixing. The separate chambers 107, 108, 109 are fluidly connected to the first chamber 101, for example, and can preferably be closed by valves.

The device 100 also comprises a detection chamber 105 which is fluidly connected to the first chamber 101 and preferably has a microarray 106 for the detection of nucleotide sequences amplified by PCR. The

Detection chamber 105 can be connected to a second opening 130 or integrated optics 130 in device 100 for optical readout or a further access or outlet. For the transportation of to the R. 371836

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Beads 131 bound target proteins in the detection chamber 105 can the

Magnet 140 is also movable between the first chamber 101 and the

Detection chamber 105 may be arranged. Alternatively, the beads 131 can be conveyed into the detection chamber 105 by rinsing. Furthermore, the detection chamber 105 can be connected to a further chamber 104 for holding detection reagents.

FIG. 2a shows an example of a target protein 10, to which a first antibody 121 and a second antibody 122 are bound at different locations on the target protein 10. The first antibody 121 has a predetermined first

Nucleic acid fragment 151 and the second antibody 122 have a predetermined second nucleic acid fragment 152. The first nucleic acid fragment 151 is in the presence of ligase, polymerase and nucleotides and the third nucleic acid fragment 153 is formed with the second

Nucleic acid fragment 152 linked, according to a procedure in a protein extension assay. In contrast to FIG. 2a, FIG. 2b shows the situation in a protein ligation assay in which the first

Nucleic acid fragment 151 in the presence of polymerase and two

unbound nucleotide strands 10, 11 and forming the third

Nucleic acid fragment 153 binds to the second nucleic acid fragment 152 via the two unbound nucleotide strands 10, 11.

FIG. 3 shows a flow diagram of an exemplary embodiment of the

Method 500 according to the invention, which can be carried out, for example, using the exemplary embodiment of the device 100 according to the invention described above.

In a first step 501 of the method 500, the biological sample is taken up in the sample chamber 111 of the device 100. In a second step 502, the sample is contacted with a first antibody 121 and with a second antibody 122, wherein the first antibody 121 and the second antibody 122 are designed to bind to the target protein at different sites and the first antibody 121 given first

Nucleic acid fragment and the second antibody 122 have a predetermined second nucleic acid fragment. For the binding of the target proteins in the sample with the first antibody 121, the sample can first enter the first chamber 101 R. 371836

WO 2020/002396 PCT / EP2019 / 066948 10 are conveyed, for example with the aid of the magnet 140 or conventional microfluidic pumps, for example membrane pumps. For this purpose, the beads 131 with the first antibodies 121 bound to them can also be upstream in the sample chamber 111 in order to be transported into the first

To facilitate chamber 101. For this purpose, the magnet 140 can preferably be moved between the sample chamber 111 and the first chamber 101. The sample can then be conveyed further into the second chamber for binding the second antibodies 122 to the target proteins of the sample. Alternatively, the target proteins immobilized by the binding to the first antibodies 121 to the solid phase, here to the beads, can also be kept in the first chamber 101, while the second antibodies 122 via fluidic processes, in particular by actuation of pumps in the device 100 in the first chamber 101 is transported for binding with the target proteins. The

Immobilization also facilitates purification or flushing of the

Target proteins to remove unwanted components. In a third step 503, a ligase 128 or a polymerase 128 is added, which binds the first when the first antibody 121 and the second antibody 122 bind to the target protein and in the presence of unbound nucleotides 125 to form a third nucleic acid fragment

Nucleic acid fragment with the second nucleic acid fragment. For this purpose, the ligase 128 or the upstream polymerase 128 upstream, for example in the third chamber 103, and the upstream unbound nucleotides 125 can be used together with those likewise in the third

Reaction components 129 upstream of the chamber are conveyed from the third chamber 103 into the second chamber 102 or into the first chamber 101. If the reaction components 129 in separate chambers 107, 108, 109

are upstream, they can be transported out of these chambers. Second chamber 102 may further include primers 127 required for ligase reaction or polymer reaction. In a fourth step 504, the third nucleic acid fragment formed is detected as detection of the target protein in the detection chamber 105 of the device 100, preferably after a polymerase chain reaction for duplicating the third

Nucleic acid fragment. The proof can preferably be via a

Transport of the reproduced third nucleic acid fragments into the

Detection chamber 105 take place via the microarray 106 arranged there. R. 371836

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Alternatively, the polymerase chain reaction can already be carried out in the

Detection chamber 105 are carried out. In a further variant, the duplication and the detection of the target proteins take place via a quantitative real-time polymerase chain reaction, the result of the real-time PCR being able to be followed in real time via the opening or optics 130 of the detection chamber 105.

Claims

R. 371836 WO 2020/002396 PCT / EP2019 / 066948 12 claims

1. device (100), in particular microfluidic device (100), for detecting a target protein (10) of a biological sample, comprising a sample chamber (111) for receiving the biological sample, a first antibody (121) and a second antibody (122), wherein the first antibody (121) and the second antibody (122) are designed to bind to the target protein (10) at different locations and wherein the first antibody (121) a predetermined first nucleic acid fragment (151) and the second antibody (122) a given second

Have nucleic acid fragment (152), further comprising one with the

Sample chamber (111) fluidically connected detection chamber (105) for detection of a third nucleic acid fragment (153) as detection of the target protein (10), the third nucleic acid fragment (153) upon binding of the first nucleic acid fragment (151) to the second nucleic acid fragment (152) upon binding of the first antibody (121) and the second antibody (122) to the target protein (10) with the addition of ligase (128) or polymerase (128) and in the presence of unbound nucleotides (125) in the sample.

2. The device (100) according to claim 1, wherein the first antibody (121) in the

Sample chamber (111) or a first chamber (101) of the device (100) is immobilized.

3. The device (100) according to claim 2, wherein the first antibody (121) to a

Solid phase (131) is bound, in particular to magnetizable or magnetic particles (131) or beads (131).

4. The device (100) according to claim 3, wherein the first antibody (121) is bound to the solid phase (131) via a protein (132), in particular via a further antibody (132). R. 371836

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5. The device (100) according to one of claims 2 to 4, wherein the first chamber (101) with the sample chamber (111) is fluidly connected.

6. The device (100) according to any one of the preceding claims, wherein the first

Nucleic acid fragment has a first nucleotide sequence which is complementary to a second nucleotide sequence of the second nucleic acid fragment.

7. Device (100) according to one of the preceding claims, wherein the first

Nucleotide sequence and the second nucleotide sequence are complementary to two further predetermined nucleotide sequences at two locations each.

8. The device (100) according to claim 7, wherein the further predetermined

Nucleotide sequences are upstream in a chamber (101, 111) of the device (100), in particular in the sample chamber (111) or the first chamber (101).

9. Device (100) according to one of the preceding claims, wherein the device (100), in particular the first chamber (101) or the detection chamber (105), is set up for carrying out a polymerase chain reaction of the third nucleic acid fragments (153).

10. The device (100) according to any one of the preceding claims, wherein the second antibody (122) in a second chamber (102) of the device (100) is upstream.

11. The device (100) according to any one of the preceding claims, wherein the device (100) comprises the unbound nucleotides (125) and preferably primers (127) and enzymes (128) for the formation of the third nucleic acid fragment (153).

12. The device (100) according to any one of the preceding claims, wherein the device (100) comprises reaction components (129) for the enzyme functions of the ligase (128) and / or the polymerase (128).

13. Method (500) for the detection of a target protein (10) of a biological sample with a device (100), in particular with a microfluidic device (100), R. 371836

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- 14 - including the steps:

• Receiving (501) the biological sample in a sample chamber (111) of the device (100).

• Contacting (502) the sample with a first antibody (121) and with a second antibody (122), the first antibody (121) and the second antibody (122) being formed, to the target protein (10) at different locations bind and wherein the first antibody (121) has a predetermined first nucleic acid fragment (151) and the second antibody (122) has a predetermined second nucleic acid fragment (152).

• Addition (503) of a ligase (128) or a polymerase (128) which forms upon binding of the first antibody (121) and the second antibody (122) to the target protein (10) and in the presence of unbound nucleotides (125) a third nucleic acid fragment (153) binds the first nucleic acid fragment (151) to the second

Nucleic acid fragment (152) causes.

• Detection (504) of the third nucleic acid fragment (153) as detection of the target protein (10) in a detection chamber (105) or in the first chamber (101) of the device (100), preferably after a polymerase-ketene reaction to multiply the third

Nucleic acid fragments (153).