METHOD FOR PERFORMING AN AMPLIFICATION REACTION IN A MICROFLUIDED DEVICE

Description

[0001] The present invention relates to a method for carrying out an amplification reaction in a microfluidic device, wherein the reaction is carried out using starting materials labeled with fluorophore and quencher and a chemical energy carrier for exciting the fluorophores by means of energy transfer. The invention further relates to a kit and a microfluidic device for carrying out such an amplification reaction. State of the art

[0002] The polymerase chain reaction (PCR) is a fast and highly sensitive method for DNA analysis developed in the 1980s. Using specific primers, a defined enzymatic reaction is performed. in vitro- DNA replication is carried out, in which the target sequence is duplicated (amplified) in several cycles. While radioactive labeling of the reaction products was initially used, fluorescence has now become the standard method for their detection. For this purpose, the reaction can be performed using starting materials labeled with a fluorophore and a quencher. To generate fluorescence, the fluorophores or fluorescent dyes are optically excited with a suitable excitation wavelength. If a quencher is located in close proximity to the fluorophore, the fluorescence is deactivated without radiation. If the fluorophore and quencher are spatially separated during the amplification reaction, the fluorescence can be measured.

[0003] For the reaction, primer chains labeled with a fluorophore or so-called TaqMan probes, which contain a fluorophore and a quencher in close proximity, can be used, for example. During the polymerase chain reaction, these chains or probes are incorporated and / or hydrolyzed, thus breaking the spatial proximity between the fluorophore and quencher, and fluorescence from the reaction products can be detected after optical excitation. The excitation light and the fluorescence emission usually have different wavelengths (so-called "Stokes shift" between absorption and emission maxima). With the aid of optical filters in a detection device, these wavelengths can be separated from each other, so that ideally a detector or detection camera only detects the light with the wavelength of the fluorescence emission. This is described, for example, in WO 2004 / 033726 A1.

[0004] GB 2 365 866 A describes the use of photoluminescent nucleic acid chains as fluorophores.

[0005] WO 2008 / 072209 A2 describes a microdevice for performing PCR analysis. A photoluminescent polymer is arranged at the bottom of an analysis chamber. This polymer can be excited to luminescence by irradiation. The luminescence wavelength corresponds to the excitation wavelength of a fluorophore used as a probe. Disclosure of the invention Advantages of the invention

[0006] The proposed method serves to carry out an amplification reaction in a microfluidic device, for example, on a lab-on-a-chip within so-called silicon microarray cells. Such microfluidic devices are already used, for example, in medical diagnostics. The amplification reaction is performed using starting materials labeled with a fluorophore and a quencher. During the amplification reaction, the fluorophore and quencher are separated, enabling the detection of reaction products and the evaluation of the amplification process by means of detectable fluorescence emission. According to the proposed method, detection is achieved by adding at least one chemiluminescent substance and evaluating the resulting fluorescence emission of the fluorophores.Unlike conventional methods, the proposed method does not require optical excitation of the sample volume to enable fluorescence readout. Instead, the excitation of the fluorophores occurs through the transfer of chemical energy using the chemiluminescent substance.

[0007] Chemiluminescence is based on the fact that corresponding chemiluminescent substances can be chemically excited to a higher energy state. This excited energy state of chemiluminescent substances is generally long-lived; that is, it is typically an energy state from which an optical transition to a ground state is forbidden, for example, due to dipole selection rules. For instance, the excited energy state might be a so-called triplet state (T1), from which an optical transition to a singlet ground state (S0) is forbidden. The present invention utilizes the relatively long-lived chemical excitability of chemiluminescent substances, since the energy from these long-lived excited states can be transferred particularly easily to other molecules, for example, to excite the aforementioned fluorophores to fluorescence emission through energy transfer.The excitation of the chemiluminescent substances themselves is achieved, for example, through an oxidation reaction with or without a catalyst. The key aspect of the proposed method is therefore that the fluorescence is not optically excited, but rather that energy is transferred to the fluorophores using chemiluminescent substances, allowing the fluorescence emission of the fluorophores to be evaluated in the absence of quenchers in the immediate vicinity. The excitation energy is thus supplied to the quenched or non-quenched fluorophores after the amplification reaction via a chemiluminescent substance. Furthermore, it is advantageous that the substances acting as energy carriers are water-soluble so that they can be used in buffer systems such as those employed for these types of reactions.

[0008] This novel combination of a chemically energy-carrying molecule with suitable fluorophores eliminates the need for optical excitation. Optical excitation for generating fluorescence is often problematic or costly, especially in microfluidic devices, as it requires homogeneous light irradiation of a defined intensity and wavelength over the largest possible area of ​​the microfluidic device. This necessitates complex and expensive optics, which are undesirable for microfluidic devices, particularly for point-of-care and / or high-throughput applications, due to cost considerations. By using chemiluminescent substances according to the proposed method for exciting the fluorophores, such complex and expensive optics can be avoided during the amplification reactions.The proposed method thus allows an optical readout method for an amplification reaction, for example for a PCR reaction and / or an isothermal amplification reaction, in microfluidic devices, without the need for an optical light source and corresponding optical devices to excite light emission.

[0009] The chemiluminescent substance can be, for example, 3-aminophthalhydrazide (5-amino-2,3-dihydrophthalazine-1,4-dione; known as luminol) and / or 3-nitrophthalhydrazide. Such chemiluminescent substances are characterized by the fact that they are excited to a specific state by the addition of energy, particularly via an oxidation reaction, and thereby emit light. Since these are long-lived excited energy states, the chemiluminescence of the aforementioned substances is comparatively weak. The wavelength or energy corresponding to the light emission (luminescence radiation) is used in the proposed method to be transferred non-radiatively to the fluorophores, thus exciting the fluorescence of the fluorophores separated by the quenchers. In principle, this energy transfer occurs independently of the luminescence radiation.

[0010] The reaction leading to molecular excitation and subsequent energy transfer is based primarily on an oxidation reaction. In this respect, the chemiluminescent substance derives its chemical excitation energy primarily from a (catalyzed) oxidation reaction. Hydrogen peroxide or carbamide peroxide (= hydrogen peroxide-urea) can be used as the oxidizing agent, so that the detection in the proposed method is preferably carried out in the presence of hydrogen peroxide. The particularly preferred chemiluminescent substance, luminol, is converted into a long-lived excited (triplet) state by oxidation with hydrogen peroxide, acting as a chemical energy carrier. This stored excitation energy can then be transferred to other, particularly longer-wavelength emitting fluorophores, thus enabling fluorescence as evidence of the amplification reaction.This energy transfer from the weakly luminescent luminol or related molecules to a strongly fluorescent molecule, such as rhodamine or yakima yellow, leads to an overall increase in light emission, which is why it is also called "chemical sensitization".

[0011] The oxidation reaction that excites the chemiluminescent substances is driven by the decomposition of hydrogen peroxide. The hydrogen peroxide can be present, for example, in the form of carbamide peroxide, i.e., a mixture of hydrogen peroxide and urea. Additionally or alternatively, alkaline conditions can be established for the detection, whereby, for example, the reaction liquid containing the chemiluminescent substance includes at least one alkaline substance. For instance, dilute sodium hydroxide or sodium bicarbonate solution can be used, which increases the solubility of the chemiluminescent substances, such as luminol, and simultaneously accelerates the decomposition of hydrogen peroxide. Alternatively, suitable buffers can be used.

[0012] The detection is particularly advantageous when performed in the presence of at least one catalyst that catalyzes the chemical reaction underlying the energy-transfer process. Preferably, the catalyst catalyzes an oxidation reaction. The catalyst may preferably be potassium hexacyanoferrate(III) and / or manganese peroxide and / or hydroquinone or related substances, e.g., catechol or resorcinol. Since some catalysts do not interact with the actual amplification reaction, in particularly preferred embodiments, such a PCR (or amplification)-compatible catalyst may already be present in the microfluidic device of the microarray cells. Hydroquinone, for example, is particularly suitable in this context. In other embodiments, enzymatic catalysts, e.g., Horseradish Peroxidase (HRP) are used.

[0013] Preferably, the at least one chemiluminescent substance is added by layering the microfluidic device (for example, the silicon microarray chip) with a reaction fluid containing the at least one chemiluminescent substance and optionally further components, such as hydrogen peroxide and optionally a catalyst. Layering the microfluidic device here means that the reaction fluid is introduced into the microfluidic device, particularly after completion of the amplification reaction, so that layering occurs above the reaction chambers of the microfluidic device. The reaction fluid here refers to the fluid in which the excitation of the chemiluminescent substances takes place, i.e., in particular, the excitation of the chemiluminescent substances.Preferably, this involves an oxidation reaction to excite the chemiluminescent substances, which can subsequently transfer their chemical energy to the fluorophores. Layering has the particular advantage that the oxidation reaction and the energy transfer to the fluorophores essentially only take place at the interface of the liquids, where the different components mix by diffusion. This allows the reaction used for evaluation to be utilized more efficiently over time, and the generated fluorescence can be detected over a longer period.

[0014] This embodiment of the proposed method therefore involves the use of a chemically excited luminescence reaction to excite the fluorescence, which is described as Readout or a detection reaction for an amplification reaction. This combination is highly advantageous for microfluidic devices, as the optical excitation, which is often difficult to achieve with microfluidic devices, is unnecessary and can be replaced by adding the appropriate reagents for the luminescence reaction. This can be easily accomplished by layering with a suitable reaction fluid after completion of the amplification reaction, in the sense of an endpoint reaction. Such layering is easily automated, making this method particularly advantageous for the use of microfluidic devices, for example in medical diagnostics, with high throughput and minimal equipment requirements.

[0015] Fluorescent dyes that emit in a longer wavelength range than the chemiluminescent substances used are generally suitable as fluorophores. This has the advantage that the interfering luminescent radiation and the fluorescent radiation of interest for the actual analysis can be easily separated by optical filters, effectively blocking out the interfering luminescent background radiation for evaluation. Rhodamine B and / or other rhodamines and / or Yakima Yellow and / or Cy-5 are particularly suitable as fluorophores, with Yakima Yellow being especially preferred. The emission maximum of Yakima Yellow is at 550 nm and extends beyond 600 nm. The absorption maximum of Yakima Yellow is at 525 nm.The maximum luminescence emission of luminol is at 425 nm and longer wavelengths, so non-radiative energy transfer to Yakima-Yellow can easily occur. However, the wavelength of the fluorescence emission from Yakima-Yellow (550 nm or more) and the wavelength of the "interfering" luminescence emission from luminol (425 nm or more) differ so significantly that they can be easily separated by an optical filter, allowing the luminescence emission from luminol to be masked for analysis.

[0016] In particularly preferred embodiments of the method, the evaluation is carried out by using at least one optical filter to separate interfering luminescent radiation from the fluorescence radiation. Advantageously, the optical filter is adapted to the respective substances used and their emission wavelengths, in particular the luminescence emission wavelength and the fluorescence emission wavelength, so that in the case of luminescent substances, the luminescence emission wavelength is filtered out. For example, an optical bandpass filter with a transmission wavelength of, say, 570 nm and / or an optical edge filter with a transmission wavelength of, say, 570 nm or longer can be used for filtering, so that in the case of luminol and Yakima yellow, the two emissions of this example can be separated with particularly high efficiency.

[0017] In a particularly preferred embodiment, the amplification reaction is an endpoint reaction, specifically an endpoint PCR or an endpoint reaction of an isothermal amplification reaction. When the procedure is carried out "at the endpoint," no Readout The detection reaction is not initiated during the amplification reaction, but rather the end of the amplification reaction is awaited before the detection reaction is started by adding the appropriate substances or by layering the chemiluminescent substances and the substances required to initiate the luminescence reaction. After the amplification reaction has completed, i.e., when the multiplication reaction in the reaction chambers of the microfluidic device has reached its endpoint, the microfluidic device can be layered with the reaction fluid containing the chemiluminescent substances and, if necessary, other substances such as a catalyst and hydrogen peroxide as an oxidizing agent. It is also possible and advantageous to have the catalyst already present in the PCR chemistry. In the latter case, PCR-compatible catalysts must be selected, i.e., substances that do not negatively affect the PCR. For example,Hydroquinone is suitable for this purpose. It generates the chemiluminescent substance, transferring its chemical energy to the fluorophores. If the fluorophores are spatially separated from their quenchers as a result of the amplification reaction, the fluorescence can be detected as evidence of the amplification. Therefore, the light emission of the fluorescence is proportional to the amount of quencher-free fluorophores, which in turn is proportional to the amount of amplification. Thus, the fluorescence light emission is proportional to the amount of products formed, allowing for an evaluation of the amplification reaction based on the fluorescence.

[0018] The invention further comprises a kit for carrying out the described amplification reaction in a microfluidic device. This kit preferably comprises two reaction mixtures, wherein the first reaction mixture contains components for carrying out the amplification reaction and the second reaction mixture contains components for carrying out the detection reaction.

[0019] The first reaction mixture primarily contains the starting materials suitable for a fluorescence readout of the reaction, which are specifically labeled with one or more fluorophores and quenchers, for example, appropriately labeled primers or probes. In addition, the first reaction mixture may contain standard substances for carrying out an amplification reaction, such as for a standard PCR reaction or an isothermal amplification reaction, e.g., buffer, nucleotides, primers, and / or DNA polymerase. These standard substances for carrying out the amplification reaction may be included optionally or, depending on the application, provided and added by the user. Furthermore, the first reaction mixture may also contain a PCR-compatible catalyst for the subsequent oxidation reaction (detection reaction).To carry out the reaction, the user may add the PCR-compatible catalyst to a standard PCR kit for the subsequent oxidation reaction. Furthermore, the initial reaction mixture may already contain at least one chemiluminescent substance, which is used to perform the detection reaction according to the proposed procedure. Luminol is particularly suitable as the chemiluminescent substance. The oxidizing agent, such as hydrogen peroxide or carbamide peroxide, is provided separately or in the second reaction mixture of the kit, as it would interfere with the amplification reaction.

[0020] The second reaction mixture of the kit provides components that preferably also contain the substances responsible for the reaction that excites the chemiluminescent substances. These are, in particular, the substances for an oxidation reaction, for example, hydrogen peroxide or carbamide peroxide as oxidizing agents, and optionally one or more catalysts for the oxidation reaction and / or suitable buffer substances, for example, to establish suitable alkaline conditions. As already mentioned, the catalyst, if PCR-compatible, can also be included in the first reaction mixture of the kit described above.Using the reaction mixtures in the kit, the amplification reaction can be carried out and evaluated as described above. Chemical energy is transferred to the fluorophores by means of the chemiluminescent substance, which then emit light (fluorescence) provided no quencher is nearby. For further details regarding the kit's features for carrying out the amplification reaction, please refer to the description above.

[0021] Finally, the disclosure includes, although not part of the invention, a microfluidic device for carrying out amplification reactions. This device is characterized in that it is configured for carrying out the proposed method. In particular, this device may be a lab-on-a-chip containing a silicon chip with a plurality of microarray cells, such as those already known for carrying out miniaturized reactions, for example, molecular diagnostic reactions in medical diagnostics. This microfluidic device is configured for carrying out an amplification reaction with a fluorescent Readout The reaction is set up using starting materials labeled with one or more fluorophores and a quencher. Unlike conventional methods, where fluorescence is excited optically, the excitation occurs through an energy-transfer reaction of chemiluminescent molecules. This transfers chemical energy to the fluorophores, leading to the emission of fluorescent radiation, provided no quencher is present in the vicinity of the fluorophore. The microfluidic apparatus used for this combined reaction is characterized, in particular, by the inclusion of a catalyst and / or an oxidizing agent required for the excitation of the chemiluminescent substances. Appropriate buffers may also be included.Typically, the chemiluminescent substances are added only after the amplification reaction is complete, in the sense of an endpoint reaction, for example by layering with a suitable reaction fluid containing the substances. For further features of this microfluidic device, please refer to the description above.

[0022] Further features and advantages of the invention will become apparent from the following description of exemplary embodiments in conjunction with the drawings. The individual features can be implemented individually or in combination with one another.

[0023] The drawings show: Fig. 1 Spectra of the absorption and emission of the fluorophore Yakima-Yellow, and Fig. 2 Emission spectrum of Luminol. Description of exemplary implementations

[0024] The proposed method provides a novel method for carrying out an amplification reaction in a microfluidic device, combining a known detection method for the amplification reaction based on fluorescent dyes with non-radiative excitation of the fluorophores by energy transfer from excited molecules. In the known fluorescence method used in connection with amplification reactions, fluorophore-labeled starting materials, such as fluorophore-labeled primer chains or TaqMan probes, are incorporated, hybridized, or hydrolyzed during the amplification reaction. These primers or probes contain one or more fluorophores and a quencher, which are immediately adjacent. As long as this configuration of fluorophore and immediately adjacent quencher exists, no fluorescence emission can occur.In particular, no fluorescence emission can occur from such primer chains or probes, each containing a fluorophore and quencher, as long as they are freely suspended in solution. Only through the incorporation of such primer chains or probes into an amplification product does either the fluorophore and quencher split and separate spatially, or the quencher detach from the fluorophore-bearing molecule. Such fluorophores can then emit fluorescence radiation upon excitation and represent a measure of the amount of amplification products formed. This method, through energy transfer to the fluorophores via a chemical reaction, eliminates the need for complex and expensive optics that were conventionally required for optical excitation in fluorescence detection reactions.

[0025] The proposed method can be carried out, in particular, for an endpoint PCR reaction or an isothermal endpoint amplification reaction in a singleplex or multiplex configuration within a silicon microarray or other microfluidic device. During the amplification reaction, the described incorporation of molecule pairs from quenchers and fluorophores and / or the hydrolysis of such starting materials occurs during the formation of the amplification products. After the amplification reaction has completed, i.e., once the amplification reaction in the array cells has essentially reached its endpoint, the microfluidic device can be overlaid with a reaction fluid containing the chemiluminescent substances. These substances can carry chemical energy for an extended period after excitation (independent of their luminescence), allowing this energy to be transferred to the fluorophores without radiation.

[0026] The reaction underlying the excitation of the chemiluminescent substances, particularly the oxidation reaction, can be accelerated by a catalyst. Potassium hexacyanoferrate(III) or hydroquinones, or other oxidation catalysts, are particularly suitable for this purpose. This catalyst can be added to the reaction mixture of hydrogen peroxide, luminol, and suitable buffers. Some catalysts can also be placed directly upstream in the array cells of the microfluidic device, provided they do not interfere with the actual amplification reaction, which is the case, for example, with many hydroquinones. Other possibilities for suitable catalysts include manganese peroxide (pyrolusite), which can also be placed upstream in the array cells, particularly in the form of a suspension. Manganese peroxide is not water-soluble and therefore does not interfere with the amplification reaction.Upon contact with hydrogen peroxide, manganese peroxide catalyzes the decomposition of the hydrogen peroxide molecules and the oxidation of chemiluminescent substances, such as luminol, to their long-lived excited form. Furthermore, manganese peroxide is a very inexpensive and harmless compound, making it particularly suitable as a solid-state catalyst for the decomposition of hydrogen peroxide and the initiation of the subsequent oxidation of luminol.

[0027] In principle, all fluorescent dyes that emit at a longer wavelength than the chemiluminescent substance used are suitable as fluorophores. For example, Rhodamine B, other Rhodamines, Yakima Yellow, or Cy-5 are suitable fluorophores that can be used in combination with luminol as the chemiluminescent substance. Fig. 1 The absorption and emission spectra of Yakima yellow are illustrated. The absorption maximum is at 525 nm and the emission maximum at 550 nm, while the emission extends significantly further at longer wavelengths, beyond 600 nm. The emission spectrum of luminol is shown in Fig. 2 As shown: The emission maximum is at 425 nm, and the emission extends to significantly longer wavelengths. Thus, the excitation energy of luminol can lead to the excitation of Yakima yellow. For the evaluation of the fluorescence emission of Yakima yellow, it is advantageous that both emissions—the interfering luminescence emission of luminol and the fluorescence emission of Yakima yellow—can be easily separated by optical filters, so that the luminescence emission of luminol does not interfere with or mask the fluorescence emission relevant for the evaluation of the detection reaction.

[0028] Conventional probes with the fluorophore Yakima-Yellow and a quencher are based, in principle, on a bridge structure that couples the quencher to the fluorophore. In a Anneal or upon attachment of such a labeled probe to a DNA strand, the bridge opens, thereby spatially separating the quencher from the fluorophore Yakima-Yellow.

[0029] The type of Readouts In the sense of an endpoint PCR or an isothermal endpoint amplification reaction, this can be implemented technically without great difficulty. While a single reaction does not allow for quantitative analysis, the "quantitative" characteristic is generally no longer necessary at the possible high multiplex levels. Due to the particular advantage of the proposed method, which eliminates the need for optical excitation of the sample volumes, even large-area silicon microarrays with a very large number of array cells and different detection reactions can be processed and read out in parallel.

[0030] In carrying out the procedure, a known amplification reaction with primers and / or probes suitable for a fluorescence- Readout The reactions are suitable for this purpose. After reaching the endpoint of the amplification reaction, the reaction mixture for the detection reaction according to the invention, which consists, for example, of water, luminol, hydrogen peroxide / urea (carbamide peroxide), and potassium hexacyanoferrate(III) as a catalyst for the reaction that excites the chemiluminescent substances, can be slowly fed into the PCR multiarray chip in the microfluidic device to gradually overlay the reaction mixture of the amplification reaction. To support the detection reaction, in particular to destabilize the hydrogen peroxide and to increase the solubility of luminol, a small amount of sodium hydroxide or alkaline buffer (e.g., sodium bicarbonate) can also be added, and suitable buffers can also be pre-stored in the microfluidic device.

[0031] The substances for the reaction that excites the chemiluminescent substances can be added directly to initiate the detection reaction or partially pre-stored in the array cells or elsewhere in the lab-on-a-chip system. A particular advantage of such lab-on-a-chip systems is that all necessary reagents can be pre-stored at a suitable location and in a suitable manner. In particular, a PCR-compatible catalyst such as hydroquinone is suitable for pre-storage in the array cells of the silicon chip, as hydroquinone does not interfere with the amplification reaction. Another particularly suitable option is the pre-storage of manganese peroxide (pyrolusite) as a water-insoluble solid catalyst in the array cells. Manganese peroxide, also being water-insoluble, generally does not interfere with the amplification reaction, which takes place in aqueous solution.Since manganese peroxide is a very efficient decomposition catalyst for hydrogen peroxide and thus for the oxidation of luminol, it is particularly well-suited as a starting point for the chemical excitation of the chemiluminescent substance, especially luminol. The excited luminol transfers its energy to the fluorophore, for example, Yakima yellow. Provided that the fluorophore Yakima yellow is spatially separated from its quencher, that is, in particular when the Yakima yellow primer or probe is bound to a DNA strand and the quencher is thereby separated, Yakima yellow can fluoresce. For reading out the fluorescence radiation, an optical filter (e.g. 570 - 580 nm) is preferably used, optionally combined with an additional edge filter that blocks shorter wavelength emission than 580 nm, in order to suppress the luminescence radiation from Lumiol that interferes with the evaluation.

[0032] For example, commercially available proprietary PCR kits from manufacturers not themselves the subject of the present invention are used as reaction approaches for carrying out PCR in the array cells. The selected PCR kit is optionally supplemented by suitable PCR-compatible catalysts and additional buffer substances to optimally adapt it to the environment of the array cells in the silicon chip. Furthermore, the PCR kit should preferably operate with Yakima Yellow or a suitable rhodamine as a detection fluorophore as described herein.

[0033] An exemplary reaction approach for layering the PCR kit after completion of the PCR in the array cells to trigger the detection reaction can include the following components and concentrations: Ultrapure water, hydrogen peroxide 0.1% - 10% (corresponding to 30 mMolar - 3 molar), preferably 0.5% - 5% (corresponding to 150 mMolar - 1.5 molar), alternatively carbamide peroxide (hydrogen peroxide - urea) 30 mMolar - 3 molar, preferably 150 mMolar - 1.5 molar, sodium hydroxide 10 mMolar - 1 molar, preferably 50 - 500 mMolar, particularly preferably 100-150 mMolar, alternatively sodium bicarbonate 50 mMolar - 1 molar, preferably 100-500 mMolar, luminol 10 mMolar - 150 mMolar, particularly preferably 50 - 100 mMolar, catalyst (hydroquinone, Horse Radish Peroxidase HRP, manganese peroxide, potassium hexacyanoferrate-III) 10 mMolar - 1 molar, preferably 50 mMolar-500 mMolar, particularly preferably 100 mMolar

Claims

1. Method for carrying out an amplification reaction in a microfluidic device, wherein the reaction is carried out using starting substances labelled with a fluorophore and a quencher and wherein a separation of the fluorophore and quencher that has taken place in the course of the amplification reaction allows reaction products to be detected, characterized in that the detection is carried out by adding at least one chemiluminescent substance and evaluating the fluorescence emission of the fluorophores that occurs.

2. Method according to Claim 1, characterized in that the chemiluminescent substance is 3-aminophthalhydrazide and / or 3-nitrophthalhydrazide.

3. Method according to either of the preceding claims, characterized in that the detection is carried out in the presence of hydrogen peroxide.

4. Method according to Claim 3, characterized in that the hydrogen peroxide is used in the form of carbamide peroxide.

5. Method according to any of the preceding claims, characterized in that the detection is performed in the presence of at least one catalyst.

6. Method according to Claim 5, characterized in that the catalyst is potassium hexacyanoferrate(III) and / or manganese peroxide and / or hydroquinone and / or catechol and / or resorcinol and / or horseradish peroxidase (HRP).

7. Method according to Claim 5 or Claim 6, characterized in that the catalyst is initially introduced into the microfluidic device.

8. Method according to any of the preceding claims, characterized in that the at least one chemiluminescent substance is added by overcoating with a reaction liquid in which the at least one chemiluminescent substance is contained.

9. Method according to any of the preceding claims, characterized in that the fluorophore used is Rhodamine B, another Rhodamine and / or Yakima Yellow and / or Cy5.

10. Method according to any of the preceding claims, characterized in that the evaluation is performed using at least one optical filter.

11. Method according to any of the preceding claims, characterized in that the amplification reaction is an endpoint reaction.

12. Kit for carrying out an amplification reaction in a microfluidic device according to Claim 1, wherein starting substances labelled with a fluorophore and a quencher and at least one chemiluminescent substance for a detection reaction are contained.