In situ cell analysis in cell culture systems
阅读说明：本技术 细胞培养系统中的原位细胞分析 (In situ cell analysis in cell culture systems ) 是由 M·塔施纳 V·洛伯 于 2019-05-07 设计创作，主要内容包括：本发明涉及一种原位方法,包括a)在包含活细胞和细胞培养基的二维或三维细胞培养系统中,测定选自由细胞表面分子和细胞外基质分子组成的组中的分子,具有以下步骤：i)提供分析物探针,所述分析物探针由结合所述分子的检测元件,以及一种或多种识别元件组成；ii)将分析物探针与细胞培养系统中的分子结合,其中所含的活细胞的生长能力基本上不会因这一步骤而受损；iii)任选地去除未结合的分析物探针；iv)释放分析物探针；v)将分析物探针转移到与细胞培养系统不同的容器中；vi)检测一种或多种识别元件；以及b)在细胞培养系统中继续进行细胞培养。(The present invention relates to an in situ method comprising a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a two or three dimensional cell culture system comprising living cells and a cell culture medium, having the steps of: i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements; ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step; iii) optionally removing unbound analyte probe; iv) releasing the analyte probe; v) transferring the analyte probe into a container distinct from the cell culture system; vi) detecting one or more recognition elements; and b) continuing the cell culture in the cell culture system.)
1. An in situ method comprising:
a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a two-or three-dimensional cell culture system comprising living cells and a cell culture medium, comprising the steps of:
i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements;
ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step;
iii) optionally removing unbound analyte probe;
iv) releasing the analyte probe;
v) transferring an analyte probe into a container distinct from the cell culture system;
vi) detecting one or more recognition elements; and
b) the cell culture is continued in the cell culture system.
2. Method according to claim 1, characterized in that the molecules are determined again in the same cell culture system after a certain period of time, preferably 1-7 days.
3. The method of claim 1 or 2, wherein the detection element is an aptamer selected from the group consisting of a nucleotide-based aptamer and a peptide-based aptamer.
4. The method of claim 1 or 2, wherein the detection element is selected from an antibody, an antibody fragment, an antibody derivative, or an antibody-aptamer conjugate.
5. The method of any one of claims 1 to 4, wherein the analyte probe contains one or more attachment elements.
6. The method according to any one of claims 1 to 5, wherein the cell surface molecule is a membrane protein or a cell wall protein, such as a receptor protein, a transporter protein, a cell-cell recognition protein, a cell matrix protein, an enzyme or a signaling protein.
7. The method according to any one of claims 1 to 5, wherein the extracellular matrix molecule is a glycoprotein, such as collagen, fibrin, elastin or vitronectin, or a glycosaminoglycan, such as hyaluronic acid, heparan sulphate, chondroitin sulphate or keratan sulphate.
8. The method according to any one of claims 1 to 7, wherein the cells in the three-dimensional cell culture are attached or immobilized on a surface, a solid three-dimensional framework or a hydrogel, or suspended in the form of cell aggregates or spheres.
9. The method according to any one of claims 1 to 8, characterized in that: steps ii) to iv) are carried out in a cell culture system in a static environment, preferably in a vessel, such as a microtiter plate or other culture dish, or under perfusion, preferably in a bioreactor, microfluidic system or a purified microtiter plate.
10. The method of any one of claims 1 to 9, wherein in step ii) the analyte probe is present in excess relative to the cell surface molecule or extracellular matrix molecule.
11. The method according to any one of claims 1 to 10, wherein the recognition element of the analyte probe in step vi) is detected by PCR, preferably qPCR, RT-PCR, digital PCR, touchdown PCR, asymmetric PCR, solid phase PCR or nested PCR, or indirectly by binding to a complementary binding element, preferably wherein the complementary binding element is photo-detectable.
12. The method of any one of claims 1 to 10, wherein the recognition element of the analyte probe comprises a luciferase, peroxidase, alkaline phosphatase or an enzyme reporter element and is detected in step vi) by a corresponding substrate in a vessel different from the cell culture system.
13. The method according to any one of claims 1 to 10, wherein the recognition element of the analyte probe in step vi) is detected by mass spectrometry, by flow cytometry or by sequencing, preferably by next generation sequencing.
14. The method according to any one of claims 1 to 13, wherein the molecule is chemically unchanged in steps ii) and v) and may be retained in a cell culture system, except for forming a complex with the detection element.
Technical Field
The present invention relates to the field of in situ analysis of cell surfaces, cell surface components or unbound cell components in cell culture systems.
Background
The standard method for characterizing animal cells is flow cytometry. In which cells or beads can be detected in small channels by means of lasers and detectors. These cells are mixed with a fluorescently labeled antibody that is capable of binding to and labeling a specific molecule on the cell surface. The cells are then analyzed in a flow cytometer based on the light scattering and the resulting fluorescent signal.
In the field of cell therapeutic products, monitoring of cells during culture is particularly important. In order to ensure and improve the safety and effectiveness of these products, it is necessary to conduct continuous tests on the starting materials, intermediates and final products. A quality control system is of critical importance in this context, since it ensures consistency of the end product per individual production process. In this context, there is still a lack of suitable in situ control methods that allow the determination of intermediates in cell culture systems without affecting the cells. For this reason, methods are needed that minimize the intervention on the culture, but still have high sensitivity and specificity for the analyte sought. The determination with flow cytometry, although meeting the current requirements of sensitivity and specificity, requires the separation of the analyte (cells) from the culture and its disposal after analysis.
Zhang et al (J.Clin.Microbiol.46(4),2008,1292-1297) describe a method for the determination of bacterial toxins, wherein the toxin is removed from the culture in the form of a supernatant and bound to a solid plate. The immobilized toxin was detected by binding to the antibody and iPCR (Immuno Polymerase Chain Reaction).
Hansen et al (BioTechniques 201456: 217-228) reviewed PCR and suggested a sensitivity enhancement by optimization of blocking and washing steps. The analyte to be determined is bound to a solid surface.
EP 2189539 relates to a method for the determination of conjugate complexes for use in immunoassays. The use of these conjugate complexes also requires the preparation of a sample and the isolation of the analyte in advance.
Malou et al (Trends in Microbiology,2011,19(6):295) relate to a method for the determination of small amounts of immobilized analyte by iPCR.
Terazono et al (Journal of Nanobiotechnology 2010,8:8) describe a method for fluorescent labeling of living cells. However, this method has the disadvantage that the fluorescent dye has to be removed by a purification treatment which changes the culture conditions and/or the subsequent further analysis results. Cells cannot be monitored without disturbing the growth conditions.
US 2012/0077714 a1 relates to a method of analysis (e.g. of cells) wherein the cells are coupled to a label and label-bound cells are separated from unbound cells using flow cytometry.
US 2008/0113875a1 relates to a method for determining one or more molecules in a sample by binding to a complex coupled to a mass tag. After the mass spectrometric tag is separated from the complex, it is detected by a mass spectrometer. The sample may be a cell lysate, a tissue section, or a body fluid.
AU2015/261546a1 relates to a method of determining molecules in a sample by binding to an aptamer complex. The aptamer binding molecule is bound to a solid phase and detected.
WO2017/075265a1 relates to a method for analyzing highly complex proteins or cellular components in individual cells in a hydrogel network, or individual separation units in cellular components, and for labeling cellular components with labeling ligands associated with nucleic acid tags. Cellular components can be tested using sequencing methods. The cells are embedded in the hydrogel prior to binding with the labeled ligand.
US2017/0016909a1 relates to a probe, composition, method and kit for simultaneous multiplexed detection and quantification of protein expression in user-defined regions of tissue, user-defined cells and/or user-defined intracellular subcellular structures.
US2017/0137864a1 relates to a cell labeling method for high resolution imaging. These cells are contacted with a detection probe that specifically binds to the molecule. The (fluorescently) labelled probe is then bound to the detection probe and detected by imaging methods.
US2017/0233723a1 relates to switchable aptamers and their use in the purification of certain ligands. Depending on the ion concentration, switchable aptamers have high or low affinity for selected ligands, such as viruses, cells or antibodies.
US2011/0136099a1 relates to a method for determining a target molecule in a sample by means of an aptamer, wherein the aptamer complex is immobilized on a solid support.
US2010/0151465a1 relates to aptamers immobilized on a solid support (e.g. microbeads) that can bind or release an analyte at a certain temperature.
WO 2016/201129a1 relates to a device for enriching cells using an aptamer coupled to beads, which binds to a specific cell surface marker, such as CD31, and which can be released from the cells after they have been isolated.
It is an object of the present invention to provide an improved method which makes it possible to determine molecules in their qualitative environment. In particular, specific molecules should be detected in cell culture systems.
Disclosure of Invention
It is an object of the present invention to provide an improved detection method which makes it possible to determine molecules in their qualitative environment. In particular, specific molecules will be detected in the cell culture system.
This object is solved by the subject matter of the present invention.
The present invention relates to a protoculture method for measuring molecules in a two-dimensional or three-dimensional cell culture system containing living cells. In particular, the method is characterized in that the cell culture can be continued even after the measurement of the molecule. In particular, one or more molecules in a particular cell culture may thus be assayed at different times.
The invention includes an in situ method comprising the steps of:
a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a two-or three-dimensional cell culture system comprising living cells and a cell culture medium, comprising the steps of:
i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements;
ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step;
iii) optionally removing unbound analyte probe;
iv) releasing the analyte probe;
v) transferring the analyte probe into a container distinct from the cell culture system;
vi) detecting one or more recognition elements; and
b) the cell culture is continued in the cell culture system.
In particular, the method is characterized in that the cells comprised in the cell culture system are not substantially impaired in their growth. Preferably, the growth of the cells is impaired by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50%. In particular, there is substantially no damage in cell growth due to a maximum reduction in cell growth of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50%. In particular, a maximum reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50% in cell growth is observed over a longer period of time than 6, 12, 24, 36 or 48 hours after the method. Preferably, the reduction in cell growth is at most 20%, even more preferably at most 10%, compared to a reference culture not treated by the method.
In particular, the present invention relates to an in situ process comprising the steps of:
a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a three-dimensional cell culture system comprising living cells and a cell culture medium, comprising the steps of:
i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements;
ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step, preferably wherein the growth of the cells is reduced by at most 20%;
iii) optionally removing unbound analyte probe;
iv) releasing the analyte probe;
v) transferring the analyte probe into a container distinct from the cell culture system;
vi) detecting one or more recognition elements; and
b) the cell culture is continued in the cell culture system.
Specifically, the present invention comprises an in situ process comprising the steps of:
a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a two-dimensional cell culture system comprising living cells and a cell culture medium, with the steps of:
i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements;
ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step, preferably wherein the growth of the cells is reduced by at most 20%;
iii) optionally removing unbound analyte probe;
iv) releasing the analyte probe;
v) transferring the analyte probe into a container distinct from the cell culture system;
vi) detecting one or more recognition elements; and
b) the cell culture is continued in the cell culture system.
Specifically, the present invention comprises an in situ process comprising the steps of:
a) determining a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule in a two-or three-dimensional cell culture system comprising living cells and a cell culture medium, with the following steps:
i) providing an analyte probe consisting of a detection element that binds to the molecule, and one or more recognition elements;
ii) binding the analyte probe to a molecule in a cell culture system, wherein the growth capacity of the contained living cells is not substantially impaired by this step;
iii) optionally removing unbound analyte probe;
iv) releasing one or more recognition elements from the analyte probe or releasing the entire analyte probe;
v) transferring the recognition element or the entire analyte probe into a container distinct from the cell culture system;
vi) detecting one or more recognition elements; and
b) the cell culture is continued in the cell culture system.
In particular, the method is characterized in that the molecules are determined again in the same cell culture system after a specific period of time. In particular, the molecules are determined again within 1-10 days, preferably within 1-7 days, even more preferably within 1-3 days. The particular time period may be hours, days, weeks, months or longer. In particular, the time period may be at least 3, 8,12 or 24 hours. The time period is preferably up to 1, 3, 6, 9 or 12 months. Even more preferably, the period of time is up to 1, 2, 3 or 4 weeks.
In particular, the method is characterized by the steps ofSteps ii) to v) may be carried out for up to 60, 30 or 20 minutes. At 37 ℃ and 5% CO2Does not cause any substantial damage to cell growth in the cell culture for a period of up to 60, 30 or 20 minutes.
In particular, the method is characterized in that the detection element is an aptamer, wherein the aptamer is selected from the group consisting of nucleotide-based aptamers and peptide-based aptamers.
In particular, the method is characterized in that the detection element is selected from an antibody, an antibody fragment, an antibody derivative or an antibody-aptamer conjugate (conjugate).
Furthermore, the method is particularly characterized in that the analyte probe comprises one or more connecting elements. In particular, the connecting element is located between the detection element and the identification element or between two identification elements. In particular, the linking element is selected from the group consisting of peptides, oligonucleotides and chemical cross-linkers. The linking element preferably comprises one or more cleavage sites or one or more photosensitive modifications. The cleavage site is preferably an enzymatically cleavable cleavage site or a photosensitive cleavage site. Thus, the linking element may be cleaved by an enzyme or under the action of light (preferably UV light).
Furthermore, the method is characterized in particular in that the cell surface molecule is a membrane protein, such as a receptor protein, a transport protein, a cell-cell recognition protein, a cell matrix protein, an enzyme, a signaling protein, or a cell membrane or a further component of a cell wall.
Furthermore, the method is particularly characterized in that the extracellular matrix molecule is a glycoprotein, such as collagen, fibrin, elastin or vitronectin, or a glycosaminoglycan, such as hyaluronic acid, heparan sulfate, chondroitin sulfate or keratan sulfate.
In particular, the method is further characterized in that the cells in the three-dimensional cell culture are attached or immobilized on a surface, a solid three-dimensional framework or a hydrogel, or suspended in the form of cell aggregates or spheres. In particular, the liquid three-dimensional cell culture may be present in a conical flask, a bioreactor, a microfluidic system or a purified microtiter plate.
In particular, the method is also characterized in that steps ii) to iv) are carried out in a cell culture system in a static environment, preferably in a vessel, such as a microtiter plate or other culture dish, or under perfusion, preferably in a bioreactor, microfluidic system or a purified microtiter plate.
In particular, the method is characterized in that, in step ii), the analyte probe is present in excess with respect to a cell surface molecule or an extracellular matrix molecule.
Furthermore, the method is characterized in particular in that the recognition element of the analyte probe in step iv) is detected by PCR, preferably qPCR, RT-PCR, digital PCR, touchdown PCR, asymmetric PCR, solid phase PCR or nested PCR, or indirectly by binding to a complementary binding element. Preferably, the complementary binding member is optically detectable.
The method is further characterized in particular in that the recognition element of the analyte probe has a luciferase, peroxidase, alkaline phosphatase or an enzyme reporter element and is detected in step v) by a corresponding substrate in a vessel different from the cell culture system. Preferably, the corresponding substrate is fluorescein, coelenterazine, ABTS (2,2' -azido-bis- (diammonium salt of 3-ethylbenzothiazoline-6-sulfonic acid)), PNPP (p-nitrophenyl phosphate), OPD (peroxidase o-phenylenediamine) or TMB (3,3',5,5' -tetramethylbenzidine).
The method is further characterized in particular in that the recognition element of the analyte probe has a fluorescent marker or is linked to a fluorescent marker. Preferably, the fluorescent marker is detectable by fluorescence microscopy or flow cytometry.
Furthermore, the method is particularly characterized in that in step v) the recognition element of the analyte probe can be detected by mass spectrometry, flow cytometry or sequencing (preferably next generation sequencing).
In particular, the method is also characterized in that the molecule remains chemically unchanged in steps ii) and v) and can be retained in the cell culture system, except for the formation of a complex with the detection element.
The invention also relates to a kit suitable for carrying out the method according to the invention. The kit may comprise one or more analyte probes suitable for the method according to the invention. For example, the kit comprises at least one analyte probe having a detection element adapted to bind to the desired analyte; a connecting element; at least one first identification element; and at least one label probe having a binding element complementary to the first recognition element; wherein the analyte probe and the label probe are provided in separate containers. The kit components described can be used stepwise for their obvious applicability in the method according to the invention.
Further details of the invention are equally applicable to all aspects of the invention.
Drawings
FIG. 1 shows an analyte probe made of an oligonucleotide with an aptamer as a detection element (1 a).
Figure 2 shows an analyte probe with an antibody as the detection element (9).
FIG. 3 shows a schematic sequence of an assay method using an analyte probe with an aptamer as the detection element. The recognition element is released by changing the binding conditions.
Fig. 4 shows a schematic sequence of an assay method using an analyte probe with an antibody as the detection element. The recognition element is released by restriction digestion (restriction digest).
Fig. 5 shows an analytical method using an analyte probe with an aptamer as a detection element and using qPCR (quantitative polymerase chain reaction) to analyze the recognition element.
Fig. 6 shows an analytical method using an analyte probe with an antibody as a detection element and using qPCR (quantitative polymerase chain reaction) to analyze the recognition element.
FIG. 7a shows indirect detection of CD105 on adMSC by aptamer probe, qPCR and melting curve analysis in 2D cell culture system, with dilution of the analyte probe in the upper panel at 1:250, 1:500 and 1: 1000. The lower graph shows the melting curve.
FIG. 7b shows indirect detection of CD105 on adMSC by aptamer probe, qPCR and melting curve analysis in 2D cell culture system, with dilution of the analyte probe in the upper panel at 1:5000 and 1: 1000. The lower graph shows the melting curve.
Fig. 7c shows the corresponding standard curves of fig. 7a and 7b with dilutions between 1:1000 and 1:1000 ten thousand.
FIG. 8a shows the results of indirect detection of CD105 on adMSCs in 2D control by qPCR, sample (E2/F2/G2-A2/B2/C2-A5/B5/C5) in triplicate, negative control (H2/D2/D2).
Fig. 8b shows the corresponding standard curve of fig. 8a with a dilution of 1:1000 to 1:100 ten thousand.
FIG. 9a shows indirect detection of CD105 on adMSCs in 3D cell culture systems by aptamer probe, qPCR, sample (A2/B2/C2-A3/B3/C3-E3/F3/G3) in triplicate, negative control (D2/D3/H3).
Fig. 9b shows the corresponding standard curve of fig. 9a with a dilution of 1:1000 to 1:100 ten thousand.
Fig. 10 shows indirect detection of CD105 on adMSC by qPCR in microfluidic chips, against 2D 96 well plates.
Detailed Description
According to the present invention, a molecule selected from the group consisting of a cell surface molecule and an extracellular matrix molecule can be assayed in a two-or three-dimensional cell culture system comprising living cells and a cell culture medium without substantially impairing the growth capacity of the living cells. In particular, cells in a two-or three-dimensional cell culture system comprising a cell culture medium can continue to grow without substantial damage from molecular assays.
Cell culture systems are complex cellular environments that are affected by living cells. Such an environment is composed of a large amount of cell matrix and nutrients as well as cell metabolites and various other components, and is complicated by the mixture of various substances. This complexity may itself interfere with the assay of certain detection molecules. In addition, the cellular metabolism itself can cause interfering side reactions.
The process according to the invention is an in situ process. The term "in situ method" or "in-culture method" refers to a method performed in an in vitro cell culture system comprising living cells. In the method according to the invention, living cells are therefore not removed from the two-or three-dimensional cell culture system comprising the cell culture medium. During the process according to the invention, the cells remain in the cell culture system. The cell culture system comprises a vessel suitable for cell culture, optionally containing a three-dimensional framework for three-dimensional cell culture, and a cell culture medium. The cells may be washed in various steps of the method, but the washing step may also be performed using a cell-compatible medium. Since the cells are treated during the method using only a cell-compatible medium, cell growth is not substantially impaired and can continue even after the determination of the molecule or molecules. Even if the molecules were measured continuously on different days, cell growth was not substantially impaired. The method according to the invention is therefore particularly suitable for monitoring the potential change of molecules over a specific period of time.
According to the invention, at least the first step of the detection, i.e.the binding of the analyte probe to the molecule, is carried out in the cell culture system (i.e.in the presence of cells and cell culture medium). The analyte probe or a portion thereof (e.g., the detection element) can be retained in a complex cellular environment without purification treatment, and the probe or the remainder thereof does not have any substantial effect on cell growth and therefore does not significantly impair cell growth. Alternatively, the analyte probe or a portion thereof may be degraded in a complex cellular environment, in particular, naturally degraded by the cell or its catabolic enzymes.
The molecules determined according to the in situ method of the invention are cell surface molecules or extracellular matrix molecules.
A cell surface molecule is a molecule associated with a cell membrane or a biological membrane of a cell compartment or organelle of a cell. There is a distinction between peripherical membrane proteins (proteins bound to the membrane surface) and integral membrane proteins, which are proteins that are integrated with hydrophobic components in the membrane bilayer, which most span the membrane bilayer as transmembrane proteins.
Extracellular matrix (ECM) is a part of animal tissue, particularly connective tissue, and is present between cells in what is known as the intercellular space. From a present day perspective, the ECM includes a large ensemble of macromolecules, which are located outside the plasma membrane of cells in tissues and organs. Glycosaminoglycans (GAGs) are long-chain polysaccharides composed of disaccharide units of certain sugars, found in large amounts in the ECM. The following should be mentioned here: hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate and keratan sulfate. In addition to hyaluronic acid, all GAGs can bind to proteins, thereby forming proteoglycans. Almost all cells have receptors that contact the ECM. Different adsorption, adapter or other adhesion proteins are usually used, which are themselves components of the ECM, interacting on the one hand with other components of the matrix and on the other hand with cell receptors.
Preferred examples of cell surface molecules are membrane proteins, such as receptor proteins, transport proteins, cell-cell recognition proteins, cell matrix proteins, enzymes, signaling proteins, or other components of the cell membrane or cell wall. Preferred examples of extracellular matrix molecules are glycoproteins, such as collagen, fibrin, elastin, vitronectin, laminin, or glycosaminoglycans, such as hyaluronic acid, heparan sulfate, or chondroitin sulfate. The cell surface molecule may also be a recombinant protein.
The analyte probe according to the invention (in the method) has a detection element adapted to bind the desired molecule, and one or more recognition elements. It is generally a compound of these two components, with or without a connecting element (linker). The chemical linkage between the two elements is preferably a covalent bond.
The analyte probe is preferably a small molecule, for example less than 50kD in size. The analyte probe may have a size or molecular weight of 50Da to 40kDa, 100Da to 30kDa or 200Da to 20kDa, or preferably 400Da to 10 kDa. In the case of nucleic acids, the detection element may consist of an oligonucleotide of 6 to 80 nucleotides in length. 10 to 60 nucleotides are preferred.
The oligonucleotide sequence may consist of deoxyribonucleotides, ribonucleotides and/or analogs thereof, and chemically modified deoxyribonucleotides or ribonucleotides. Preferably, the recognition element consists of an oligonucleotide sequence of 8 to 100 nucleotides (e.g., 16-20 nucleotides), however, the sequence may be longer.
The detection element is a molecule (or a molecular component of an analyte probe) that can bind to the selected molecule. This binding reaction is a specific reaction to obtain a corresponding specific signal. There are various bonding mechanisms for the binding of molecules, preferably complex formation reactions or ionic interactions. The non-covalent bonds are reversible and the linkage can be broken by changing the binding conditions, for example by increasing the temperature or increasing the salt concentration, or by adding chemicals/detergents which cause bond denaturation. Thus, in step iv) of the method according to the invention, the entire analyte probe is released from the molecule, preferably by changing the binding conditions. Covalent reactions are also possible. Suitable bonds on the molecule are ligand-ligand bonds or ligand-receptor bonds. In particular, in the case of cell surface molecules, the corresponding ligands of these cell surface molecules can be used as detection elements.
Typically, the detection element may be a peptide, protein or nucleic acid.
The universal detection element comprises an antibody or antibody fragment or derivative having an antigen binding portion. Aptamers are similar universal detection elements. Antibodies (generally including fragments or derivatives thereof) and aptamers to virtually any molecule can be obtained and used in accordance with the present invention.
In particular embodiments of the methods discussed, antibodies, antibody fragments or derivatives can be used as detection elements.
Preferred antibody fragments or derivatives are single chain antibodies or antigen binding domains thereof, Fab, Fv, F (ab)2、Fab'、F(ab')2、scFv、scfc、VHH. The antibodies may be of the IgG, IgA, IgD, IgE, IgM, IgW and IgY class, or fragments or derivatives derived from these antibodies. The antibody may be a polyclonal antibody or a monoclonal antibody.The bond of the antibody to the other element of the analyte probe may be a covalent bond or formed by a complex-for example, the aptamer may bind to the antibody, for example being linked to the remaining element of the analyte probe at the Fc portion. The covalent bond is preferably via a lysine residue of the antibody. Covalent bonds to the antibody may also be formed by common cross-linking agents such as EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide). Here, the carboxyl group is attached to a primary amine group, such as a lysine residue. In other embodiments, the functional group of the oligonucleotide sequence may also bind to a primary amine (-NH)2) Mercapto (-SH), carbonyl (-CHO) or other commonly used groups. Bonds via photoreactive groups are also possible. According to a specific embodiment, an antibody or antibody fragment with low binding affinity is used as the detection element. The lower the affinity of a protein (e.g., an antibody) for a ligand, the greater the Kd value. In particular, the advantage of using an antibody with a high Kd value and a low affinity for the target molecule is that the analyte probe (comprising the detection element and the recognition element) can be more easily separated from the target molecule. This can be done, for example, by changing the ion concentration in the medium or by applying a shearing force on the detection element by perfusion.
In a preferred embodiment, aptamers are used as detection elements and/or recognition elements. For example, the use of aptamers for detection methods is disclosed in WO 2009/012420A 1, WO 2005/113817A 2, DE 102010038842A 1, WO 2012/130951A 1, EP 1918372A 1, Baumstmler et al (Letters in Applied Microbiology 59,2014: 422-. In particular, slow rate modifying aptamers (somamers, Baumstummler et al, supra) are preferred.
According to a particular embodiment, the aptamer is peptide-based and is referred to as a peptidyl aptamer. In this case, aptamers consist of short (5-20) amino acid sequences that are linked to a small, very stable protein backbone to form aptamers. This structure provides a particular conformation that increases the likelihood of specific binding to the desired molecule. Generally, peptide-based aptamers can be considered as shrinking immunoglobulin T cell receptors. Examples of peptidyl aptamers and their use are Adnectin a, a bispecific molecule intended for use in cancer; anticalin, an aptamer against cytotoxic T lymphocytes CD 152; or DARPin, a VEGF-a inhibitor used in the treatment of various eye diseases (e.g., macular edema). (Sergey Reverdatto, David S.Burz, and Alexander Shekhtman, Peptide Aptamers: Development and Applications, Curr Top Med chem. 2015; 15(12): 1082. Burz 1101.)
According to another specific embodiment, the aptamer is nucleotide based and is referred to as a nucleotide based aptamer. Preferred nucleotide-based aptamers are short single-stranded DNA or RNA oligonucleotides between 25-90 bases in length. These oligonucleotides are capable of binding to specific molecules and exhibit a suitable 3D structure. Aptamers can bind to proteins, bacterial toxins, low molecular weight substances (such as antibiotics and amino acids), cells, cell surface molecules, cell matrix molecules, and viral particles. Their dissociation constants are in the picomolar and nanomolar range. This means that they can bind as strongly as antibodies.
According to a particular embodiment, a combination of an antibody and an aptamer (in particular, such as an antibody-aptamer conjugate) is used as the detection element. Antibody-aptamer conjugates include in particular antibodies, antibody fragments or antibody derivatives, which are preferably covalently bound to synthetic oligonucleotides (e.g. aptamers). The synthetic oligonucleotide is preferably 40, 50, 60, 70 or more bases in length. Synthetic oligonucleotides are preferably used as recognition elements. After the detection element has bound to the desired target molecule, the recognition element is preferably isolated by digestion with restriction enzymes (e.g., EcoRI, HindIII, etc.) and transferred to a different container along with the culture supernatant. Analysis can then be performed similarly to the case of aptamer-based detection elements.
Another possible way to bind antibodies, antibody fragments or derivatives to aptamers is by using Fc-specific aptamers as detection elements. The constant Fc part of an antibody, antibody fragment or derivative can thus be bound non-covalently and isolated again, for example by varying the ion concentration in the culture medium. This allows different antibodies, antibody fragments or derivatives obtained from cells of different animal species to be used in e.g. multiplex assays, since each animal species incorporates a unique Fc part into its antibody.
An example of a covalent bond of the detection element to the molecule is a photoreactive reaction, e.g., by a photoaptamer. The detection element may be covalently bound to the molecule when exposed to light. For example, photoaptamers, which typically have photoreactive groups, are described in U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, U.S. Pat. No. 6,291,184, and U.S. Pat. No. 6,458,539.
Aptamers can be produced using SELEX. In molecular biology, the abbreviation SELEX (Systematic Evolution of Ligands by amplification) is understood as a combinatorial approach for the directed Evolution of oligonucleotide strands, such as single-stranded DNA or RNA. These oligonucleotide strands can act as ligands to specifically bind to a selected target.
The library of base sequence sequences is screened for the most strongly binding ligand to the desired molecule by creating a large random library of oligonucleotides with different base sequences and performing exponential enrichment by systematic evolution. To do this, candidate aptamers are mixed with immobilized ligands and unbound aptamers are washed away. In a preferred embodiment of the method in question, the candidate aptamer is incubated with the cell in a cell culture medium in a first SELEX round or in a further SELEX round. Thus, in a complex cellular environment comprising a cell culture medium, aptamers are selected that specifically bind to the corresponding cell and the molecule to be determined. An advantage of selecting in a complex cellular environment is that the selected aptamers are not negatively affected in their binding specificity by large amounts of cellular material, nutrients, cellular metabolites and various other components or cellular metabolism. It is these aptamers that are capable of specifically binding to defined molecules in this complex cellular environment, which are referred to as cell culture-selected aptamers according to the invention.
After this step, the candidate aptamers are additionally subjected to negative selection and protein selection. After the above-mentioned rounds of selection, these aptamers are selected in further rounds to select aptamers that specifically bind to the defined molecule. Here, the defined molecules are bound to a solid support (preferably magnetic beads) and then selected. In negative selection, selection is performed in the complete absence of molecules. The purpose of this step is to remove from the library aptamers that bind only to the solid support and not to the molecule. All aptamers bound to the solid support remain on it, preferably the remaining aptamers are subjected to further selection.
Finally, what remains are candidate aptamers with high affinity (binding strength) for the target molecule. The individual characteristics of aptamers are high chemical stability, low immunogenicity, high specificity and affinity and the ability to specifically influence protein-protein interactions. Aptamers are generally not biosynthesized, but are chemically synthesized. This makes aptamer production less expensive. This synthesis allows for a variety of modifications, such as the addition of fluorescent reporter molecules or affinity tags. Preferred aptamers or modified aptamers are photoaptamers and spiegelmers. The analyte probe, the detection element and/or the first recognition element may consist of a nucleic acid (e.g. DNA or RNA or a mixture thereof). DNA is preferred because of its higher stability. Nucleotides may be modified, for example, methylated, arylated, acetylated. Modified nucleotides are, for example, 2 '-fluoro, 2' -methoxy and/or 2 '-amino modified nucleotides, 2' -fluoro, 2 '-methoxy and/or 2' -amino modified ribonucleotides and deoxyribonucleotides. Another possibility is LNA (locked nucleic acid) or PNA (peptide nucleic acid), especially LNA or PNA at the part which should not be cleaved or should be prevented from being cleaved.
The detection element preferably binds to the molecule with high affinity, e.g., with an association constant (dissociation constant) Kd of 10-4、10-5、10-6、10-7、10-8、10-9、10-10、10-11Or 10-12Or lower, and ranges between these Kd values.
According to a further embodiment, the detection element binds to the molecule with a low affinity, e.g. with a Kd above 10-3、10-2、10-1、101、102、103Or 104Or a higher order,and ranges between these Kd values. In particular, when the detection element is an antibody or antibody fragment, the detection element binds to the molecule with low affinity.
The recognition element of the analyte probe may be a chemical unit capable of subsequently generating a signal. It does not have to carry a (bear) signal (even if this is an option) but can generate a signal from it. The identification element is therefore usually a label (also called tag or label according to english terminology) which is subsequently bound, this binding serving to generate a signal. Reasonably, no or only a minor amount of signal generating components remains in the cellular environment after isolation (by transfer or degradation in the cellular environment) via step iv). A typical recognition element is an oligonucleotide having a characteristic sequence which is subsequently recognized for detection, for example by binding to another substance, a solid phase or another recognition element. The recognition element may also be a peptide that can be specifically recognized for detection. The signal is then generated similarly to the oligonucleotide. Also, any other form of chemical substance that can be subsequently specifically recognized for detection is possible. An analyte probe according to the invention may comprise one or more recognition elements. In particular, the analyte probe may comprise up to 5, 10, 20 or more recognition elements.
The method may further comprise a washing step or a step for short-term denaturation of the analyte probes or immobilization of components on the beads or wells of the microtiter plate. These steps can also be used to separate the identification elements.
Preferably, the molecule is chemically unchanged through steps ii) and iv) except for forming a complex with the detection element, and preferably may remain unchanged in the cell culture system. This applies in particular to cells or cell components, but generally to any molecule that can still be utilized by the environment and the cells contained therein.
The method of the invention comprises the step of binding the molecule to an analyte probe via its detection element in a two-or three-dimensional cell culture system comprising living cells and a cell culture medium. This is also step ii). Two-dimensional or three-dimensional cell culture systems comprising living cells and cell culture media are the above-mentioned complex cellular environments. Preferably, the complex cellular environment comprises living cells that are not substantially damaged or are only minimally affected by the step. In particular, cell growth should not be substantially compromised. In particular, in this step, the cells are not isolated but are assayed in situ and retained in the cell culture system, that is, the cells are retained in their culture medium throughout the analysis. For example, the cells may be cultured in advance for 1 day or longer, or 2, 3, 4 days or longer. After step ii), the cells are preferably further cultured, for example for 1, 2, 3, 4 days or more, before they are subjected again to step ii) of the method under consideration within a specific time. Preferably, the cell culture system is not altered except for the change of the medium and any passage.
In particular, the cell culture system comprises a vessel and a culture medium, without containing specific atmospheric components. Thus, in step ii) of the method in question, the cells may be removed from the incubator, typically with atmospheric conditions in the incubator in order to promote the best possible growth. The atmospheric conditions for the optimal growth of animal cells possible are preferably 35 to 38 ℃, particularly preferably about 37 ℃, and 0 to 10% CO2Particularly preferably 3-6% CO2。
The analyte probe may be present in excess relative to the molecule.
A cytocompatible liquid, in particular a cell culture medium, is preferably used in the method according to the invention. Steps ii) to v) are preferably carried out in the liquid. The liquid is used as part of a complex cellular environment. It may be a medium suitable for growing or caring for cells. The defined cell culture medium is a group of ingredients based on amino acids, carbohydrates, inorganic salts and vitamins. Typically contain at least calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and sodium phosphate. The vitamins that are usually present are at least folic acid, nicotinamide, riboflavin and B12. In addition, the cell culture medium may further contain FCS (Fetal Bovine Serum (Fetal Calf Serum)) or FBS (Fetal Bovine Serum)). Preferred cell culture media are MEM, alpha-MEM, DMEM, RPMI, and variants or modifications thereof.
The cell culture system may have cells cultured in two or three dimensions. A typical two-dimensional method involves culturing a cell suspension in a cell culture dish, where cells can attach and propagate. This method is basically simple, inexpensive and widely used worldwide, but cells in this culture mode are physiologically different from cells in vivo. For this reason, new concepts for three-dimensional cultured cells are being intensively studied and developed. For example, cells can be cultured in special bioreactors with a three-dimensional matrix (scaffold). In another variant, they are transformed using special microtiter plates, hanging drops, or into cell microbeads/spheres. In three dimensions, in many cases previously, cell-specific characterization was not possible or could only be performed at the end of the culture period using special, time-consuming analytical methods. With the present invention, in situ detection methods can now be performed in three-dimensional cell culture systems.
The three-dimensional cell culture system refers to the micro-structural three-dimensional culture of cells under in vitro conditions. The culture or cells thereof should be spatially oriented. This occurs primarily in the form of hydrogels made from structural proteins (e.g., fibrin, collagen, gelatin (polymethylmethacrylate or matrix gels), and solid scaffolds (e.g., polystyrene, polylactic acid, or other chemicals). in a three-dimensional environment, many cell lines form spheres whose diameter increases over time as cells are embedded "WAVE"/cell bag bioreactor or perfusion bioreactor. In the bioreactor, the culture medium may be delivered unidirectionally, bidirectionally, pulsated, homogeneously or randomly.
The cell may be selected from prokaryotic cells, eukaryotic cells, cells with a cell wall, in particular plant cells and fungal cells, cells without a cell wall, in particular animal cells. Preferably, the cell is a mammalian cell, particularly preferably a human cell, but may also be a non-human cell.
The cells may be stem cells, such as pluripotent, multipotent or unipotent stem cells, differentiated tissue cells, blood cells or lymphocytes. These cells may be from an immortalized cell line, such as a tumor cell line. It is also possible to culture the cells in such a way that differentiation occurs. Thus, cells may also change the degree of differentiation. Preferably, this should be determined by the culture conditions (e.g., growth or differentiation factors) and not be affected by the binding of the analyte probes.
Living cells are preferably attached or immobilized on a surface or an extracellular three-dimensional framework. However, the cells may also be present in suspension in the form of cell aggregates or spheres (or in combination therewith). The cells are preferably adhesion-bound cells. Alternatively, the cells may be non-adhesively bound. Particularly preferred are cells in suspension. Examples of 3D culturing are mostly non-adhesive binding, culturing in gels, especially hydrogels, such as fibrin or matrix gels. The surface may also be other cells, such as feeder cells, which affect the growth of the cells to be analyzed.
The method according to the invention further comprises an optional step of removing unbound analyte probes (step iii). Whether this step is performed depends on the type of further detection method to be determined which binds to the analyte probe or its first recognition element. Optional steps which are possible here are, for example, specific cleavage of only the molecule-bound recognition element or, conversely, of only the recognition element of the free (non-molecule-bound) analyte probe and their quantitative determination in order to determine the amount of reduction by the other analyte probe bound to the molecule. However, the simplest method is usually to remove unbound analyte probes by washing or degradation so that they are no longer detectable at further stages of the method. For example, degradation may be by enzymatic degradation, for example by nucleases or peptidases/proteases.
The essential steps of the method according to the invention are releasing the analyte probe or one or more recognition element from the binding of the molecule and transferring the analyte probe or one or more recognition element into a container different from the cell culture system (step v), which helps to achieve the goal of minimal interference with the cell culture system. The release may be performed in any number of steps. The release is typically performed by breaking a chemical connection, e.g. between the detection element and the first identification element. The free recognition element is then transferred to another container, thereby separating it from the cell culture system in which the cells can continue to grow. This allows the cells to continue to grow without interference from the detection method.
In a preferred embodiment, the analyte probe is released from the molecule by changing the binding conditions. In particular, the release is carried out by varying the salt concentration. The salt concentration in the complex cellular environment can be temporarily increased or decreased without causing substantial damage to the growth of the cells. The salt concentration of the cell culture medium is preferably increased to 300-600mM NaCl, more preferably to 400-500mM NaCl, or to 500mM NaCl. In order not to cause substantial damage to cell growth, the salt concentration should not increase or decrease significantly for more than 15 minutes, preferably not more than 10 minutes.
The transfer may be by affinity binding of, for example, the first recognition element or another part of the analyte probe. To this end, for example, a capture unit bound to a solid phase (e.g., a solid bead) can bind the recognition element. Using the capture unit or solid phase, the recognition element can be easily removed from a complex environment and transferred to a new container. In the new container, the recognition element may be separated from or left on the solid phase. This transfer may also be performed by any other step. The beads are preferably microbeads on which are in turn binding units, such as oligonucleotides complementary to the separation moiety (the first recognition element or a part thereof). Depending on the method, either a detection element (in particular an aptamer (buffer change/consumption)) or a recognition element is dissolved. These beads are preferably not modified like Minx (Luminex) beads or Viracoded (Vera-Code) beads. Beads of the same size can be used, in the case of multiplex assays, where different recognition elements bind to different molecules via different aliquots. Different target/bead populations can be analyzed simultaneously by physical separation, e.g., in individual wells of different test tubes or well plates.
The method according to the invention further comprises the step of detecting the recognition element, step vi). This can be done in any way, now irrespective of the cell growth conditions, in a vessel different from the cell culture system. Possible detection methods are, for example, binding to a labeled probe (labeled probe) and detecting the label. The label probe may have a binding member complementary to the recognition element and thereby bind to the recognition element. The tag preferably provides a quantifiable signal. For example, the signal may be, for example, an optical signal (in particular fluorescence) or a radioactive signal.
The method according to the invention is also particularly suitable for determining the concentration of any molecule, in particular of cell surface molecules and extracellular matrix molecules, when the target molecule is directly labelled. Indirect detection is performed when the concentration of the dissolved/cleaved first recognition moiety corresponds to or is proportional to the concentration of the target molecule/protein. Preferably, but not exclusively, the solubilised/cleaved first recognition moiety consists of a nucleic acid sequence and may be achieved by any conventional standard nucleic acid assay method.
In a particularly preferred embodiment of the method, the analyte probe is free of a linking element between the detection element and the first recognition element. In step iv), the analyte probe can be released completely from the binding of the molecule, so that the entire analyte probe with the one or more recognition elements is separated from the binding of the molecule.
Other embodiments are analyte probes having a linking element. In step iv), the linking element may be cleaved to separate the recognition element from the bonding of the molecule, such that the detection probe portion having at least one recognition element is separated from the bonding of the molecule. The linking element may have a cleavage site, in particular an enzymatic cleavage site. Suitable cleavage sites may be in the form of peptides or oligonucleotides, for example. Chemical modifications are also possible, for example oligonucleotide backbones with photosensitive modifications. The linking element may also be bound to the detection element by a photosensitive chemical cross-linking agent. This photosensitivity enables cleavage by exposure to light in step iv).
The use of 2 or more recognition elements enables discrimination between 2 or more target molecules. The detection element a and the recognition element a are bound to the molecule a, and the detection element B and the recognition element B are bound to the molecule B. After the separation step (e.g., by changing the salt concentration), the analyte probe, which consists of the detection element and the recognition element, remains in the supernatant and can be transferred. The transferred analyte probe can be determined on the basis of the recognition element by means of a detection device, for example a labeled probe with a signaling unit. For example, different recognition elements (A or B) of different molecules to which they bind can be determined using different labeled probes (A 'or B') as recognition elements or by spatial separation. This allows multiple measurements to be made in parallel (multiplexed). In one embodiment, up to 25, up to 50, up to 100, up to 200, up to 500, up to 1000, and up to 10000 analytes can be analyzed simultaneously.
In the case of oligonucleotides, the recognition element sequence may be selected such that no self-dimers (autodimers) nor dimers with the detection element or the linking element occur. If dimerization cannot be avoided, binding to other elements can be prevented by RNA sequences complementary to the recognition element. For example, the RNA sequence can be degraded with a ribonuclease prior to detection.
In the case of oligonucleotides, the invention may also be described as: a method for assaying molecules having the steps of: providing an analyte probe made of an oligonucleotide, the analyte probe having as a detection element an aptamer adapted to bind to a desired molecule and having at least one first recognition element; binding the analyte probe to the molecule via the aptamer; removing unbound analyte probes; releasing the analyte probe from the binding of the molecule by changing the salt concentration; binding the recognition element of the released analyte probe to a labeled probe, which is specifically bound for this purpose and which comprises an oligonucleotide and a sequence thereof (as a binding element) that hybridizes to the recognition element; optionally removing recognition elements not bound to the labeled probes; detecting the recognition element bound to the labeled probe.
After transfer of the analyte probe together with the recognition element, the recognition element can be determined by the labeled probe or by an intermediate step of binding a second recognition element, which in turn is bound to the labeled probe, in a new container. Such a second recognition element may be provided or manufactured in situ, for example by PCR directly at the first recognition element.
Preferably, the signal of at least the first recognition element is quantitatively amplified by performing quantitative PCR. Thus, the amount of the generated second secondary identification element is a multiple of, or related to, the amount of the first identification element. In this embodiment, the first recognition element is preferably a PCR primer and the secondary recognition element is a TaqMan labelled probe. The label probe bound to the first recognition element or the second recognition element, or any other recognition element associated with the first recognition element, is used to generate a signal. For example, the signal can be used to generate colorimetric, fluorescence or mass spectrometric assays, as well as for assays by pattern-based detection, capillary electrophoresis, chromatography, HPLC, or sequencing (e.g., NGS).
The recognition element of the analyte probe is preferably an oligonucleotide. In step vi) the detection may be by PCR, preferably qPCR, RT-PCR, digital PCR, touchdown PCR, asymmetric PCR, solid phase PCR or nested PCR, or indirectly by binding to a complementary binding element, wherein preferably the complementary binding element (e.g. a labelled probe) is photo-detectable. The first recognition element of the analyte probe may be provided with a signal substance. For example, it may have a luciferase, peroxidase or alkaline phosphatase or some other enzyme reporter element. The detection in step vi) can be carried out by a corresponding substance in the container which is different from the environment or the sample. In step vi), the recognition element of the analyte probe is preferably determined by mass spectrometry, flow cytometry or sequencing, preferably Next Generation Sequencing (NGS).
According to another embodiment, in the case of oligonucleotide probes, the recognition element can be transferred into a suitable hybridization buffer and standard hybridization methods can be performed. To ensure efficient binding of the two strands, for example, the oligonucleotide solution may be incubated at 37-60 ℃, preferably 37-50 ℃ for 2 hours or less, and each individual bead population placed in a single well of a microtiter plate. Additionally or subsequently, an intercalating fluorescent dye or a complementary fluorescently labeled detection strand is added. The solution is then assayed in a well plate reader or flow cytometer or similar device. In one embodiment, the assay can be used to simultaneously analyze up to 25, up to 50, up to 100, up to 200, up to 500, up to 1000, and up to 10000 recognition elements for different molecules. Alternatively or in addition to spatial separation, measurement of different molecules can also be performed without separation of the recognition element (multiplexing), so that different complementary samples can be immobilized on the bead population. The released recognition element may be bound to a different labelled probe. To distinguish between these molecules, different signals are used, such as fluorescent dyes with different absorption/emission spectra, which are coupled to complementary labeled probes.
In another embodiment, beads of different sizes are used, each size containing a unique label probe that binds to a different recognition element of the corresponding molecule to which the detection element binds. Spatial separation is not necessary. Depending on the method, the first identification element is used (for example the detection element itself (in the case of a binding change method)) or cut for the determination. The determination can be performed by flow cytometry, and the signal, e.g. the fluorescence signal, is obtained as a function of the size of the beads (see US2010/279888a 1). Preferably, up to 5 or more target molecules are simultaneously analyzed by these differently sized microbeads. By spatially separating these beads, e.g. in different Eppendorf tubes or well plates, it is possible to simultaneously analyze a variable number of molecules.
In one embodiment, the assay is performed by a DNA hybridization chip. For this purpose, in particular oligonucleotide analyte probes are used. According to this method, the recognition element is hybridized to the sample at a single spot on the chip. In this method, after isolation of the first recognition element, it is transferred into a suitable hybridization buffer and subjected to standard hybridization methods. To ensure the most efficient binding of the two strands, the oligonucleotide solution may be incubated with the chip, for example, at 37-60 deg.C, preferably 37-50 deg.C, for 12 hours. Thereafter, an intercalating fluorescent dye or a complementary fluorescently labeled probe can be added. The chip was washed and read in a fluorescence scanner. The result is the intensity of the fluorescent signal in the image, which can be quantified using software. In one embodiment, up to 25, up to 50, up to 100, up to 200, up to 500, up to 1000, and up to 10000 identification elements can be simultaneously analyzed by using the chip.
In the above method, particularly in the case of using an aptamer as a recognition element, the recognition element may be amplified before detection. For example, the use of aptamers or recognition elements can be accomplished by PCR prior to hybridization to the chips, beads or well plates.
In one embodiment, detection is by molecular beacon probes. These probes consist of nucleotide sequences, the ends of which are complementary to each other. Having a fluorophore (e.g., EDANS) at one end and a quencher (e.g., DABCYL) at the other end, which is capable of quenching the fluorescent signal. The middle portion consists of a sequence complementary to any portion of the analyte probe. Under normal conditions, the sequence is in the form of a hairpin structure, with two complementary ends forming a stem and the middle forming a loop. The fluorescent signal is thereby quenched by the quencher. The analyte probe having the first recognition element is released from the molecule.
In related or other embodiments, it may still bind to a component of the initial probe. After release of the analyte probe having the first recognition element, it is transferred to a molecular beacon probe. The recognition element binds to the oligonucleotide sequence of the molecular beacon and prevents the formation of a hairpin structure. When the quencher is no longer able to quench the fluorescent signal, the result is a fluorescent signal. In a further embodiment, the detection is performed during or after PCR amplification of the released analyte probe or recognition element.
In one embodiment, the recognition element is detected and quantified by qPCR after isolation. qPCR refers to performing PCR reactions under controlled conditions to determine molecular concentrations.
In one embodiment, the concentration is determined by TaqMan PCR. The TaqMan probe is based on a complementary sequence of a first recognition element, e.g. on an aptamer or the recognition element itself, and contains a fluorophore, e.g. 6-carboxyfluorescein, at the 5 'end and a quencher, e.g. 6-carboxytetramethylfluorescein, at the 3' end. The TaqMan probe binds to the recognition element, generating a signal during PCR. To this end, the polymerase replicates the sequence of the first recognition element, wherein the 5'-3' exonuclease activity releases the fluorophore and the quencher is no longer able to quench the signal. The signal increases with the number of amplification rounds and the PCR product depends on the number of cycles and the concentration of starting material.
In another embodiment, the concentration is determined by adding an intercalating fluorescent dye during amplification. This dye (e.g., SYBR-Green) produces a stronger fluorescent signal in the presence of double stranded DNA than in the presence of single stranded DNA. During the PCR process, double stranded DNA is formed, which in turn increases the signal of the dye. The signal intensity depends on the number of cycles and the concentration of the starting material.
In another embodiment, the concentration may be determined by adding fluorescently labeled nucleotides during amplification. The fluorescent dye (e.g., fluorescein), is coupled to the incorporated uracil during amplification. The signal intensity depends on the number of cycles and on the concentration of labeled uracil and therefore also on the concentration of the starting substance.
In another embodiment, detection is by capillary gel electrophoresis. A unique mass tag (mass tag) having a fluorophore is attached to the first recognition element. The labeled first identification element is then transferred to an instrument, wherein the labeled first identification element can be identified based on the mass label and quantified by a fluorescent signal.
In another embodiment, the detection is performed by a mass spectrometer. For example, a special mass tag may be attached to the first recognition element by an enzyme. Because the labels are of different qualities, the distinction is made based on the quality. This method does not require dye labeling.
In another embodiment, the detection is by High Performance Liquid Chromatography (HPLC). On the one hand, the method enables the target molecule to be isolated, and on the other hand, it allows the target molecule to be identified and quantitatively determined by means of the determination standard. In this case, the substance to be detected is mixed with an eluent (mobile phase) and eluted from a separation column (stationary phase).
In particular, the invention may also be provided in the form of a kit having all or some of the components listed.
Examples
The invention is further described by the following examples, but is not necessarily limited to these specific embodiments of the invention.
Example 1: structure of analyte probe
As shown in fig. 1, the analyte probe may consist of:
1. from the left, the aptamer (1a) may comprise one or two recognition elements (3a, 5a) as recognition sequences as a single strand.
2. From the left, here shown as single strand. These recognition sequences may be attached to the 5 'or 3' end of the aptamer. A linking element (2a) (RNA or DNA) may be provided between the aptamer and the recognition sequence. The linking element may be single stranded (2. left start, 2a) or double stranded (3. left start, 2a and 2 b).
3. Starting from the left, 2b shows one strand of the connecting element complementary to strand 2 a. In a double-stranded embodiment, the recognition element and the detection element may be present on different strands or on the same strand. Double-stranded (double Stranddness) allows the use of double-stranded specific restriction enzymes to cleave the ligation element. In addition, qPCR identification sequences (qPCR tags, 4a) may be provided.
4. From the left, optionally, the further connecting element (6) has a peptide sequence or a chemical cross-linker.
5. It is also possible, starting from the left, for the aptamers to have recognition elements (7) as inducible signaling units, for example enzymes which can signal under the influence of a substrate. Enzymes are, for example, luciferase, peroxidase, alkaline phosphatase. 2b/3b/4b/5b represents a strand (DNA or RNA) complementary to 2a, 3a, 4a, 5 a. The comb structure shows hybridizable nucleic acids or hybridized nucleic acids, provided that the counterpart is shown.
Table 1 contains a list of exemplary elements of the analyte probes according to the invention shown in fig. 1.
Table 1: elements of the analyte probe of FIG. 1
In an exemplary embodiment of the analyte probe, provided according to fig. 2 is an antibody (9) rather than an aptamer.
1. From the left, the antibody is attached to the rest of the analyte probe via a linker (8). Other elements can be seen in fig. 1.
2. From the left, alternatively the antibody may be bound by an aptamer (9a) that specifically binds to the antibody, for example by the Fc portion of the antibody.
3. In the embodiment from the left and to the right in fig. 2, the antibody is bound to the enzyme recognition element (13) via the linking elements 2a, 2 b. Suitable enzymes are, for example, luciferase, peroxidase, alkaline phosphatase or others.
Table 2 contains a list of exemplary elements of the analyte probe according to the invention shown in FIG. 2
2a
Connection label
2b
Complementary strand to the ligation tag
3a-5a
Primer sites
4a
qPCR label (identification element)
8
Crosslinking agent
9
Cell-specific antibodies (recognition elements)
9a
Aptamers, specific for the Fc part of antibodies
10
Enzyme: such as luciferase, peroxidase, alkaline phosphatase
Table 2: elements of the analyte probe in FIG. 2
Example 2: process flow
Fig. 3 and 4 schematically show the process of the method according to the invention. From top to bottom, from left to right, in the direction of the arrow: cells were cultured in 2D or 3D. The analyte probe (with antibody detection element in fig. 3; as aptamer in fig. 4) binds to the cells via the detection element. The recognition element is released in fig. 3 by changing the buffer conditions and thus interfering with the binding to the cell, in fig. 4 by the endonuclease. The cells can then be further cultured (lower left). The recognition element is now isolated from the cell and transferred to another container and submitted for analysis, e.g. by PCR and amplification assays, colorimetry, by hybridization with labeled probes, capillary electrophoresis, mass spectrometry, HPLC, NGS. Fig. 5 shows a process flow using an analyte probe with an aptamer 1a as a detection element bound to a cell surface molecule (15). The probe also has primer binding sites (3a and 5a) as recognition elements. The analyte probe is released from the cell surface molecule by changing the buffer (pH and/or ionic strength). The analyte probes released from the cell surface molecules are transferred to a new container and the probes are detected by PCR. Here, forward primer 3a and reverse primer 5b bound to primer site 5a, and the first generation amplificate (and each subsequent generation) contains site 3b, site 3b binding to forward primer 3a for further reaction. Polymerase (18) is shown. The labeled probe 1a binds to a spot on the PCR product corresponding to the aptamer, whereby the aptamer, and thus the initially bound aptamer, can also be quantitatively determined by qPCR.
Fig. 6 shows an analyte probe with an antibody (9) as detection element, the antibody (9) being bound to the linking elements (2a, 2b) by a linker group, the linking elements (2a, 2b) in turn being linked to a recognition element (3a), such as a forward primer sequence, a qPCR tag (4a) and a binding site for a reverse primer (5a) (during detection the qPCR tag can be amplified and detected by PCR using two primer binding sites). The analyte probe binds to a cell surface molecule (15). Under the action of the endonuclease, the recognition elements (primers and PCR tag) were cleaved, transferred to another vessel, and determined by qPCR. Primers 5b (reverse primer) and 3a (forward primer) were used for PCR using polymerase (18). In a first step, the cleaved recognition elements can be determined using labeled probes 4a (fluorescently labeled qPCR probes) and thus the cells to which the analyte probes bind indirectly.
Example 3: aptamer development
To select suitable aptamers as detection elements, a DNA aptamer library (triple biotechnology) was selected, which consisted of a random 40 base sequence and bilaterally defined primers. The first two rounds of SELEX were performed directly on adipocyte-derived mesenchymal stem cells (adMSC/zemer heschel (Thermofischer)), followed by 10 rounds of SELEX on His-tagged CD105 protein (SinoBiological). The aptamer library was amplified by PCR (Biomers For Primer/bioRevprimer, Solis BioDyne Hot-Start FIREPOL DNA polymerase) before the first round of SELEX For cells, and then incubated with streptavidin beads (Promega). After incubation, the duplex was denatured with 0.1M NaOH for 5 minutes, transferred to a new edbend (Eppendorf) tube, and the pH adjusted to 7 with 0.1M HCl.
In the first round of SELEX, admCs were washed in T-75 flasks (Greeny organisms (Biogreiner)) using 5ml Alpha-Mem (Sigma Aldrich) without FCS (P3). Aptamer libraries were diluted with 3ml Alpha-Mem without FCS, transferred to cells and incubated at 37 ℃ for 30 min. Then, the cells were washed 3 times with 5ml Alpha-Mem without FCS, and in order to release any aptamers that were not washed away, these aptamers were incubated with dmem (roth) +500mM NaCl (sigma-aldrich) for 10 minutes at 37 ℃. The eluate was transferred to 15ml of Furkon (Falcon) and the volume was reduced using a 3k Amicon ultracentrifugal column (Merck) according to the manufacturer's instructions. The eluate was amplified by PCR and a second round of SELEX on cells was performed as described above. In rounds 3-12, cells were replaced with HIS-tagged CD105 protein. The CD105 protein was coupled to magnetic anti-HIS beads (promegate) prior to undergoing the SELEX round. In the first 6 and 12 rounds, negative selection was performed before hatching. For this, the aptamer library was incubated with "empty HIS beads" in Alpha-Mem without FCS for 20 minutes on a rotator, and then magnetically separated from each other. The supernatant (unbound aptamer) was then incubated with HIS-CD105 (coupled with anti-HIS bead) on a rotator for 30 minutes at room temperature. After incubation, the construct was washed 3 times with Alpha-Mem without FCS and the aptamers were eluted with DMEM +500mM NaCl for 10 min. The aptamer eluate was concentrated using a 3k Amicon ultracentrifugation column and then used for PCR. Rounds 3-8 of SELEX are directed to 2. mu.g of CD105 protein (32pmol) and rounds 9-12 are directed to 1. mu.g of CD105 protein (16 pmol). Agarose electrophoresis was performed at 80V for 30 and 40 minutes at different times as a control for SELEX. The PCR products were visualized on a 3% agarose gel (50ml TAE (Sigma) +1g agarose (Longsha (Lonza)), and under a lamp by PEQGreen (Peqlab)). After the 12 th round of SELEX, final PCR was performed, and the obtained aptamer fraction was prepared for cell culture experiments.
Example 4: detection of CD105 in two-dimensional cell culture with aptamers of different concentrations
Adipose cell-derived mesenchymal stem cells (adMSC) in Tx175 flasks at Alpha-Mem (sigma) + 10% FCS (sigma) + HEPES 12mM (sigma) + MOPS 12mM (sigma) + NaBi 5mM (sigma) + 0.5% PenStrep (sigma), no CO at 37 ℃. (sigma)2The incubator of (1). During the culture, the medium in the cell culture is replaced with fresh medium every 2 to 3 days. At 75-80% confluence, cells were passaged and transferred to a new Tx175 flask. adMSC was passaged to P2 and seeded at a cell number of about 10000C in wells of a 96-well plate. As a negative control (non-specific binding), individual wells without cells but with medium were incubated.
After 2 medium changes over a week, cell assays were performed using the analyte probes prepared in example 3. The analyte probe used comprised the aptamer selected in example 3 that specifically binds to CD105 (detection element) and a qPCR tag as recognition element.
Treated aptamers were serially diluted with FCS-free Alpha-Mem at 1:250, 1:500, 1:1000, 1:5000, 1:10000 (FIG. 7). Next, the dilution was heated to 95 ℃ for 2 minutes, followed by 4 ℃ for 5 minutes. At the end of 5 minutes, it was returned to room temperature. Cells were washed with 200 μ L FCS-free Alpha-Mem, and 50 μ L of each aptamer dilution was added in triplicate to cells and blank wells (negative control/non-specific binding). The plates were incubated at 37 ℃ for 30 min. The cultures were then washed 2 times with 150. mu.L FCS-free Alpha-Mem and incubated with 50. mu.L DMEM +500mM NaCl per well for 10 min. The supernatant was transferred to a microtiter plate for storage, sealed with foil paper and stored at 4 ℃ until qPCR assay. qPCR was performed using 25. mu.L batches consisting of 5. mu.L SybrGreen 2xMastermix (Sigma), 1. mu.L FrPrimer (10 pmol/. mu.L), 1. mu.L RevPrimer (10 pmol/. mu.L), 17. mu.L ddH2O, 1. mu.L of eluent. After 10 minutes of start at 95 ℃, the cycling program (40 times) was carried out for 15 seconds at 95 ℃,1 minute at 48 ℃, 30 seconds at 72 ℃, and then the melting curve analysis was run for 1 minute at 95 ℃, 30 seconds at 48 ℃ and 30 seconds at 95 ℃ (fig. 7).
Melting curve analysis showed peaks for the desired PCR product (CD105 aptamer), however, starting at a dilution of 1:5000, more and more unwanted DNA fragments were found, which is why a dilution of 1:500 of the eluate was chosen for the experiment.
According to the PCR results, 1:500 dilutions of the eluate were amplified by means of PCR (Frprimer/BiotRevprimer) as analyte probes for further experiments and prepared with streptavidin beads for further assays.
Example 5: detection of CD105 in two-and three-dimensional cell cultures in continuous time
Adipose cell-derived mesenchymal stem cells in Tx175 flasks at Alpha-Mem + 10% FCS + HEPES 12mM + MOPS 12mM + NaBi 5mM + 0.5% PenStrep, without CO at 37 ℃2Cultured in an incubator. During the culture, the medium in the cell culture is replaced with fresh medium every 2 to 3 days. At 75-80% confluence, cells were passaged and transferred to a new Tx175 flask.
Plates were prepared for cell culture according to the manufacturer's instructions before transferring the cells to ALVETEX (Reprocell Co.). ALVETEX plates were wetted with 100. mu.L 70% ethanol for 5 min and then washed 2 times with 1 xPBS. In the final step, 100. mu.L of Alpha-Mem containing FCS are transferred to wells of a microtiter plate and the plate is transferred to 37 ℃.
After passage 4, cells were transferred to ALVETEX and MIMETIX using a Handystep pipette (Brand) at a cell count of approximately 10000/well.
As a negative control (non-specific binding), a separate well containing no cells but medium was used. As a further visual control, 2D cell cultures were performed in parallel in 96-well plates.
The medium was replaced every 2-3 days with fresh Alpha-Mem + 10% FCS. Three-dimensional cell cultures were assayed on days 4 (T1), 10 (failure of T2 by qPCR), 17 (T3) and 24 (T4) of several days in sequence, and two-dimensional cell cultures were assayed on days 4 (T1), 11 (T2) and 18 (T3) of several days in sequence. After each assay, the cells were further cultured until the end of the experiment (day 24 (3D), day 18 (2D) and beyond).
The analyte probes used comprised the CD105 aptamers selected in example 3, which specifically bound to CD105 (detection element), and a qPCR tag as recognition element.
The analyte probe was diluted 1:500 with Alpha-Mem without FCS and heated to 95 ℃ for 3 minutes, then cooled on ice for 5 minutes, and then used at room temperature. Cells were washed 1 time with 150. mu.L Alpha-Mem without FCS, 50. mu.L of aptamer solution was transferred to each well and incubated at 37 ℃ for 30 min. Excess aptamer was then removed by washing 3-4 times with 150 μ L Alpha-Mem without FCS. Bound aptamers were eluted with 50 μ L DMEM +500mM NaCl for 10 min and transferred to microtiter plates for storage. The microtiter plates were sealed with foil and stored at 4 ℃ until qPCR. qPCR was performed using 25 μ L batches consisting of 12.5 μ L of 2-fold GreenMastermix LowRox (merry organisms (biotechrabbitt)), 1 μ L FrPrimer (10pmol/μ L), 1 μ L RevPrimer (10pmol/μ L), 5.5 μ L ddH2O, 5 μ L eluent. After 3 minutes at 95 ℃ start-up, a cycling program (40 times) was run at 95 ℃ for 15 seconds, at 48 ℃ for 30 seconds, at 72 ℃ for 30 seconds, followed by a melting curve analysis run at 95 ℃ for 1 minute, at 48 ℃ for 30 seconds, and at 95 ℃ for 30 seconds (fig. 8 (two-dimensional) and fig. 9 (three-dimensional)).
Melting curve analysis showed that the PCR product of the CD105 aptamer was constant in all three measurement points in both the two-dimensional cell culture system (fig. 8a) and in the three-dimensional cell culture system (fig. 9a) and provided conclusions about the purity of the PCR product.
The metabolic activity of the cells was monitored during the experiment using the culture medium supernatant. For this purpose, the culture supernatant was directly evaluated visually for the color change of the pH indicator in the culture before the first measurement and one day after the measurement. This evaluation shows that the metabolic activity of the cells is only slightly impaired or not impaired at all according to the method of the invention.
Example 6: microfluidic control
Cell culture
The cell concentration was 106Frozen tubes of individual cells/mL mesenchymal stem cells isolated from fat (adMSC) were thawed out of a liquid nitrogen tank and then seeded in T-flasks. Cells were incubated at 37 ℃/5% CO2Incubate, change culture medium every 2-3 days. When about 50% confluency, passaging and reseeding with PBS and Agkistrodon enzyme.
In the microfluidic system experiments, cells were cultured in parallel in 96-well plates. For this, 50000 cells/100. mu.L of growth medium were transferred to a defined number of wells. In the case of microfluidic chips, the cell suspension is applied to the chip using a microscope and a syringe. After confirmation of the presence of cells in the chip, it was incubated for half an hour for attachment and then the flow of growth medium was initiated through the system. In both systems, cells were grown confluently and the medium was replaced with growth medium containing 5% FBS. For negative controls, wells and microfluidic chambers containing growth medium with only 5% FBS without cells were used.
Comparison: 96-well plate
Before starting, the solution was preheated to room temperature. The CD105 aptamer probe was then diluted to 1:500, heated to 95 ℃ for 3 minutes, and then placed on ice until further processing. Cells were washed with 150. mu.L of binding buffer, treated with 50. mu.L of diluted probe, and incubated at 37 ℃ for 30 minutes. The cells were then washed 6 times with binding buffer and 50 μ L of elution buffer was added. After incubation at 37 ℃ for 10 min, 2. mu.L aliquots were taken as samples for the qPCR assay.
Microfluidic PDMS chips:
prior to use of the microfluidic chip, analyte probes are prepared as described above. The analyte probe used comprised the CD105 aptamer selected in example 3, which specifically binds to CD105 (detection element), and a qPCR tag as recognition element.
The time for addition of each solution was as follows:
10 min-binding buffer
30 min-dilution of analyte probes
60 min-binding buffer
10 min-elution buffer
50 μ L of the eluate was collected and used as a sample for qPCR testing using 2 μ L.
qPCR
MasterMix for qPCR was prepared (see table 3) from which 23 μ L was transferred to wells of qPCR plates. The sample was then added, the qPCR plate sealed with tape and transferred to a qPCR instrument. The procedure described in table 4 was entered and started.
Table 3: qPCR
Step (ii) of
Temperature of
Time
Hot start
95℃
15 minutes
Circulating for 40 times
95℃
15 seconds
48℃
1 minute
72℃
30 seconds
95℃
1 minute
48℃
30 seconds
Table 4: qPCR program
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