Method and device for analysing a biological sample
The method and device integrate PCR amplification and fluorescence detection to analyze multiple analytes, including DNA and RNA, in a single device, addressing the limitations of traditional immunoassays by enabling simultaneous detection of low and high-molecular-weight targets.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ROBERT BOSCH GMBH
- Filing Date
- 2025-12-12
- Publication Date
- 2026-06-18
Smart Images
Figure EP2025086820_18062026_PF_FP_ABST
Abstract
Description
[0001] R. 414811
[0002] - 1 -
[0003] Description
[0004] title
[0005] Method and apparatus for a bi' sample
[0006] The present invention relates to a method for analyzing a biological sample. The present invention also relates to a device configured to carry out the method.
[0007] State of the art
[0008] Competitive immunoassays enable the detection of analytes in a biological sample that can bind to an antibody. For this purpose, the antibody is immobilized on a carrier. It is loaded with an artificial antigen, which, for example, contains a fluorescent dye. This artificial antigen has a lower affinity for the antibody than the analyte. When the carrier is brought into contact with the analyte, the analyte displaces the artificial antigen from the antibody, as described, for example, in Luttmann, W., Bratke, K., Küpper, M., Myrtek, D. (2014). Quantitative Immunoassays. In: Der Experimentator: Immunologie. Experimentator. Springer Spektrum, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-41899-04.If the carrier is monitored by fluorescence spectroscopy, it can be seen that this displacement manifests itself in a decrease in a fluorescence signal, since the loading of the antibodies immobilized on the carrier with the artificial antigen, which has the fluorescent dye, decreases when it is displaced by the analyte.
[0009] Such a competitive immunoassay is described in WO 93 / 11146 A1. It is a competitive binding method for the quantitative determination of a polynucleotide analyte containing at least one purine nucleotide. A receptor enzyme possesses a signal-generating substrate. R. 414811
[0010] - 2 - which may be labelled with a fluorescent marker. If this marker is displaced by the receptor enzyme, the fluorescent signal of the fluorescent marker decreases, thus enabling quantitative determination of the analyte.
[0011] R. 414811
[0012] - 3 -
[0013] Disclosure of the invention
[0014] The method for analyzing a biological sample comprises providing a biological sample containing at least one target antigen to be detected by the analysis. This biological sample is preferably a liquid biological sample that may contain different sample species. In particular, the biological sample may be a cell culture supernatant, urine, or blood. Furthermore, at least one antibody is provided, which is immobilized on a carrier material. The carrier is understood to be the entirety of the carrier, the antibodies immobilized thereon, and optionally, antigens bound to the antibodies. An artificial antigen is bound to the antibody. This artificial antigen carries a nucleic acid, in particular single-stranded DNA, bound to it. The target antigen has a higher affinity for the antibody than the artificial antigen with the nucleic acid bound to it.The artificial antigen can be, in particular, an analogue of the target antigen, similar to the target antigen in its chemical structure. Alternatively, the target antigen itself can be used in purified form. The nucleic acid can be attached to the artificial antigen using known methods, especially covalently or via free COOH or -OH groups. Attaching the nucleic acid can reduce the affinity of the artificial antigen for the antibody, which is particularly advantageous when using the purified target antigen or an analogue of the target antigen as the artificial antigen. When the carrier is brought into contact with the biological sample, and thus the antibody comes into contact with the target antigen, the artificial antigen is released from the antibody because the target antigen, due to its higher affinity for the antibody, displaces it.The nucleic acid of the released artificial antigen is then amplified. This can be done, for example, using a polymerase chain reaction (PCR), which is a known method. Detection of the amplified nucleic acid allows for the identification of the target antigen. This identification can be performed either qualitatively or quantitatively, for example, via quantitative real-time PCR (qPCR). R. 414811 correlates with this.
[0015] - 4 - advantageously the strength of the detection signal, particularly in a (quantitative) PCR, with the extent of displacement of the artificial antigen by the target antigen.
[0016] The target antigens can be those typically detectable by immunoassays, particularly competitive immunoassays. These can include smaller target antigens relative to the size of the antibodies used, such as hormones (e.g., sex hormones), as well as proteins (e.g., peptide hormones), enzymes, or cell surface markers. Preferably, the method can also be used to detect cells as target antigens, such as blood cells or tissue cells.
[0017] The method and the device presented below can be configured to detect various target antigens in parallel. For this purpose, different antibodies, each with a specific binding to a specific artificial antigen and a specific target antigen, are arranged, for example, in different areas of the carrier.
[0018] This method provides a means of detecting the target antigen that is based not on an analysis of the carrier, but instead on an analysis of the released artificial antigen. Therefore, it is not necessary for the artificial antigen to contain a fluorescent dye. However, in one embodiment of the method, the artificial antigen may contain a fluorescent dye in addition to the nucleic acid. This allows for detection of the target antigen not only by detecting the amplified nucleic acid, but also by detecting a change in the carrier's fluorescence. The result of detecting the amplified nucleic acid can then be correlated with the result of detecting the change in fluorescence.
[0019] Competitive immunoassays based solely on fluorescence analysis can be used either to detect low-molecular-weight target antigens, such as steroid hormones or peptides, or to detect R. 414811
[0020] - 5 - High-molecular-weight target antigens can be used, which can bind to an antibody. In each case, it is necessary to set up the competitive immunoassay for either low-molecular-weight or high-molecular-weight antigens. However, such a competitive immunoassay is not suitable for the detection of DNA and / or RNA. The present method, on the other hand, makes it possible to analyze a biological sample that contains DNA and / or RNA in addition to at least one low-molecular-weight target antigen. This can be amplified together with the nucleic acid of the released artificial antigen, for example, in a multiplex PCR. Subsequently, the amplified DNA and / or RNA can be detected together with the amplified nucleic acid of the released artificial antigen, even if it is present in the biological sample only in low concentrations. Thus, the method enables cross-species analysis.For example, cells, nucleic acids, and proteins can be detected together and the result read out. This method replaces several separate measurement methods and allows for faster results with less sample material.
[0021] This is achieved in particular by releasing the DNA and / or RNA from cells through cell disruption and then combining this cell disruption product with the released artificial antigen before they are subjected to amplification, preferably together or alternatively separately. If the nucleic acid is already present in free form in the biological sample, this step is omitted, for example, when detecting free DNA in blood. Whereas previously it was necessary to provide a first device to detect low-molecular-weight components of a biological sample and a second device to analyze cells contained in the biological sample for the presence of different genes or gene segments, these two analysis steps can now be implemented in a single device using the present method.Only the cell disruption and the contacting of the antibody with the biological sample should preferably be spatially separated within the device so that the antibody is not exposed to the lysis reagents used for cell disruption. R. 414811.
[0022] - 6 -
[0023] A device for analyzing a biological sample is therefore set up to carry out the steps of the procedure. It can, in particular, be designed as a microfluidic device, for example as a microfluidic cartridge.
[0024] For this purpose, it preferably comprises a first sample preparation chamber in which an antibody immobilized on the carrier material is arranged, to which an artificial antigen is bound and upstream, carrying a nucleic acid tag. The device also comprises a second sample preparation chamber configured to perform cell disruption. The cell disruption can be carried out, in particular, by a disruption method selected from the group consisting of heat denaturation, ultrasonic treatment, chemical lysis, enzymatic lysis, and combinations thereof. For this purpose, the second sample preparation chamber can, in particular, comprise at least one element selected from a heating / cooling element, an ultrasonic lance, or upstream lysis reagents.
[0025] The first sample preparation chamber is connected to the second sample preparation chamber, in particular by means of a channel.
[0026] The first sample preparation chamber preferably has at least one light source for emitting excitation light and at least one sensor for detecting fluorescence light. This embodiment of the device is advantageous if, in addition to detecting the target antigen by detecting the amplified nucleic acid, detection by detecting a change in the fluorescence of the support is also required.
[0027] In one embodiment of the device, it is provided that it has a first sample inlet leading into the first sample preparation chamber and a second sample inlet leading into the second sample preparation chamber. This embodiment of the device can be used when it is already known that the biological sample to be examined contains only either cells to be detected or low-molecular-weight components to be detected. In this case, only one of the R. 414811
[0028] - 7 -
[0029] The sample inputs are filled, and a reaction takes place in only one of the sample preparation chambers. Alternatively, a separation device can be installed upstream of both sample inputs, into which the sample is introduced. This separation device can, for example, include a filter that allows liquid and low-molecular-weight components to pass through while capturing cells. In this way, the sample can be divided into two fractions using the separation device, one fraction being introduced into the first sample input and the other fraction into the second sample input.
[0030] In another preferred embodiment of the device, it has only a single sample input. This is configured to split a biological sample between the first sample preparation chamber and the second sample preparation chamber. This embodiment of the device can be used when the biological sample is to be examined for both cells and low-molecular-weight components. It is particularly advantageous when only a small sample quantity is available, which is to be examined for as many components as possible in a single process run.
[0031] These embodiments of the device make it possible, particularly when the device is implemented as a microfluidic device, to switch the method flexibly depending on the target analyte.
[0032] Furthermore, it is preferred that the device has at least one amplification chamber. This chamber is connected to the two sample preparation chambers in such a way that a fluid stream from the first sample preparation chamber can be combined with a fluid stream from the second sample preparation chamber in the amplification chamber. This combining can either take place directly in the amplification chamber, or the fluid streams can be brought together in a line before an inlet of the amplification chamber, so that they flow into the amplification chamber together. This allows both artificial antigens and the nucleic acids bound to them from the first sample preparation chamber and the DNA and / or RNA of lysed samples to be conveyed. R. 414811
[0033] - 8 -
[0034] Cells from the second sample preparation chamber are transferred to the amplification chamber so that they can be amplified together there, for example by PCR.
[0035] In the amplification chamber, reagents for PCR, such as primers and probes for the PCR reaction, are preferably stored dry. Alternatively, these can be stored in liquid form in separate storage chambers and are pumped in to start the reaction.
[0036] Finally, the device preferably includes a readout chamber configured to detect amplified nucleic acids. The amplified nucleic acids from the amplification chamber can be introduced into this readout chamber. In particular, the readout chamber includes a DNA array.
[0037] Brief description of the drawings
[0038] Exemplary embodiments of the invention are shown in the drawings and are explained in more detail in the following description.
[0039] Figure 1 schematically shows the release of artificial antigens in one step of a process according to an embodiment of the invention.
[0040] Figure 2 shows a flowchart of an embodiment of the method according to the invention.
[0041] Figure 3 schematically shows elements of a device according to an embodiment of the invention.
[0042] Figure 4 schematically shows elements of a device according to another embodiment of the invention.
[0043] Exemplary embodiments of the invention R. 414811
[0044] - 9 -
[0045] In an embodiment of the method according to the invention, shown in Figure 1, an embodiment of the device according to the invention is configured to automatically detect two different natural target antigens 11, 12 in a biological sample, which is, for example, a patient sample such as urine, blood, sputum, or a swab. The target antigens are, for example, hormones, in particular sex hormones, or enzymes. For detection, as is generally the case with competitive immunoassays, a carrier 20 is provided on the surface of which complementary antibodies 21, 22 to the two antigens 11, 12 are arranged. Prior to the detection reaction, the antibodies 21, 22 are coated with artificial antigens 31, 32. Each of the artificial antigens 31, 32 has a single-stranded DNA strand 41, 42 specific to the artificial antigen 31, 32.The artificial antigens 31, 32 further differ from the natural target antigens 11, 12 in that, at least when DNA strand 41, 42 is bound, they each exhibit a lower affinity for their respective antibody 21, 22 than the natural antigen 11, 12. For example, the artificial antigens are also target antigens, but in purified form, or analogues of the target antigens. Finally, the two artificial antigens 31, 32 optionally exhibit two different fluorescent dyes. In a first step 51, the liquid biological sample containing the two natural antigens 11, 12 is passed over the carrier 20. In this process, the target antigens 11, 12 displace the artificial antigens 31, 32 from the antibodies 21, 22, thereby releasing the artificial antigens 31, 32, while the specific single-stranded DNA strands 41, 42 remain bound to the artificial antigens.In a further step 52, the artificial antigens 31, 32, together with the specific single-stranded DNA strands 41, 42 bound to them, are washed away along with the remaining liquid sample and a wash solution containing a buffer salt. Subsequent nucleic acid amplification, for example, quantitative real-time PCR (qPCR), allows the detection of the specific single-stranded DNA strands 41, 42 and thus indirectly the displacement of the artificial antigens 31, 32 by the target antigens 11, 21 57. R. 414811.
[0046] - 10 -
[0047] If the artificial antigens 31 exhibit fluorescent dyes, a supplementary detection method 57 can be performed as follows: While the biological sample is passed over the carrier 20, the carrier is irradiated with excitation light, and the fluorescence intensity of the fluorescent dyes of the artificial antigens 31, 32 on the carrier 20 is recorded using a camera 53. As the artificial antigens 31, 32 are increasingly displaced by the antibodies 21, 22, the intensity of the fluorescence decreases. Since the two artificial antigens 31, 32 exhibit different fluorescent dyes, a separate fluorescence intensity can be determined for each of the artificial antigens 31, 32 at a separate wavelength of the fluorescent light.
[0048] As shown in Figure 2, an embodiment of the method according to the invention enables the detection of cells, for example blood cells or tissue cells, containing DNA 61 and RNA 62, as well as proteins 63, for example peptide hormones or enzymes, surface markers 64, for example surface receptors or structural proteins, and low-molecular-weight analytes 65, such as sex hormones, contained in the biological sample 60. For example, this sample might be a urine sample, which typically contains cells, hormones, and proteins. The proteins 63, surface markers 64, and low-molecular-weight molecules 65 exhibit antigens, which can be target antigens 11, 12 to be detected in the method. For this purpose, they are subjected to process steps 51 and 52. Furthermore, preferably, a fluorescence change of the support 20 is detected 53 if the artificial antigens 31 contain fluorescent dyes.Target analytes based on DNA 61 and RNA 62, which are enclosed in cells, are released, for example, by means of a lysis reagent during cell lysis 54. This DNA 61 and RNA 62 is then combined with the artificial antigen 31, 32 and subjected to amplification 55, for example, by PCR. In this process, both the nucleic acid strands of DNA 61 and RNA 62 and the nucleic acids 41, 42 of the artificial antigens 31, 32 are amplified. The RNA 62 is transcribed into cDNA beforehand. The amplified nucleic acids are then read 56. This can be done, for example, using a DNA array, with the reading being carried out, for example, by chemiluminescence. Finally, the R. 414811.
[0049] - 11 - the obtained data were evaluated to perform a qualitative and quantitative detection 57 of the target antigens 11, 12 as well as the DNA 61 and the RNA 62. This utilizes the fact that for each target antigen 11, 12 bound to an antibody 21, 22, a nucleic acid 41, 42 was supplied for amplification 55. If the artificial antigens 31 exhibit fluorescent dyes, the result of monitoring the fluorescence change 53 of the carrier 20 can be used to validate the detection 57 of the target antigens 11, 12.
[0050] A first embodiment of the device used here is shown in Figure 3. This embodiment has a first sample input 71 for a sample containing proteins 63, surface markers 64, and / or low-molecular-weight molecules 65. A second sample input 72 is provided for a biological sample 60 containing cells with DNA 61 or RNA 62. A sample introduced into the first sample input 71 is directed into a first sample preparation chamber 73 and subjected there to process steps 51 to 53. A sample introduced into the second sample input 72 is directed into a second sample preparation chamber 74 and subjected there to process step 54. The prepared samples from the two sample preparation chambers 73 and 74 are then combined in an amplification chamber 75 and amplified there. Subsequently, they are directed into a readout chamber 76, which, for example, contains a DNA array.The device further comprises reagent reservoirs 77a-c from which reagents can be fed into the two sample preparation chambers 73, 74 and into the amplification chamber 75. This embodiment of the device can be used to introduce a biological sample 60 into only one of the two sample inputs 71, 72 if it is already known that it is to be analyzed only for either DNA 61 and RNA 62 or for proteins 63, surface markers 64, and low-molecular-weight molecules 65. However, it can also be used for parallel analysis of all sample components 61-65 if the sample is manually divided and introduced into the two sample inputs 71, 72.
[0051] Figure 4 shows a second embodiment of the device according to the invention. This differs from the first embodiment in that R. 414811
[0052] - 12 - that instead of the two sample inputs 71, 72, it has only a single sample input 77. This is configured to distribute a biological sample entered therein to the two sample preparation chambers 73, 74. This embodiment of the device according to the invention can be used when a biological sample 60 is to be distributed to all potential
[0053] Sample components 61 to 65 are to be examined.
Claims
R. 414811 - 13 - Claims 1. Method for analyzing a biological sample (60), comprising the following steps: Providing a biological sample (60) containing at least one target antigen (11, 12), Providing at least one antibody (21, 22) immobilized on a carrier (20), to which an artificial antigen (31, 32) is bound, the artificial antigen carrying a nucleic acid (41, 42), wherein the target antigen (11, 12) has a higher affinity for the antibody (21, 22) than the artificial antigen (31, 32) with the nucleic acid (41, 42) bound to the artificial antigen (31, 32), Contact (51) of the antibody (21 , 22) with the target antigen (11 , 12), wherein the target antigen (11 , 12) displaces the artificial antigen (31 , 32) from the antibody (21 , 22) (52), Amplifying (55) the nucleic acid (41, 42) of the released artificial antigen (31, 32), and Detection (57) of the target antigen (11 , 12) by detection (56) of the amplified nucleic acid (41 , 42).
2. Method according to claim 1, characterized in that the artificial antigen (31, 32) further comprises a fluorescent dye and, upon detection (57) of the target antigen (11, 21), the detection (57) of the amplified nucleic acid (41, 42) is made plausible by detecting (53) a change in the fluorescence of the carrier (20).
3. Method according to claim 1 or 2, characterized in that the biological sample (60) contains, in addition to the target antigen (11, 12), DNA (61) and / or RNA (62), which is preferably amplified together with the nucleic acid (41, 42) of the released artificial antigen (31, 32) (55), wherein detection of the amplified DNA (61) and / or RNA (62) together R. 414811 - 14 - by detecting (57) the amplified nucleic acid (41 , 42) of the released artificial antigen (31 , 32).
4. Method according to claim 3, characterized in that the DNA (61) and / or RNA (62) is released from cells by a cell disruption (54) and the product of the cell disruption (54) is combined with the released artificial antigen (31, 32) before they are jointly subjected to amplification (55).
5. Device for analyzing a biological sample (60) configured to perform the steps of a method according to any one of claims 1 to 4.
6. Device according to claim 5, characterized in that it has a first sample preparation chamber (73) in which an antibody (21, 22) immobilized on a carrier (20) is arranged, to which an artificial antigen (31, 32) is bound, which carries a nucleic acid (41, 42), and a second sample preparation chamber (74) which is configured to carry out cell disruption (54) therein.
7. Device according to claim 6, characterized in that it has a first sample input (71) which opens into the first sample preparation chamber (73) and a second sample input (72) which opens into the second sample preparation chamber (74).
8. Device according to claim 6, characterized in that it has a sample input (77) configured to split a biological sample (60) between the first sample preparation chamber (73) and the second sample preparation chamber (74).
9. Device according to one of claims 6 to 8, characterized in that it has at least one amplification chamber (75) which is connected to the two sample preparation chambers (73, 74) in such a way that a fluid flow from the first sample preparation chamber (73) can be combined in the amplification chamber (75) with a fluid flow from the second sample preparation chamber (74). R. 414811 - 15 - 10. Device according to claim 9, characterized in that it has a selection chamber (76) which is configured to detect (56) amplified nucleic acids (41 , 42) therein.