Method for Carrying Out an Amplification Reaction in a Microfluidic Apparatus

Abstract

In an amplification reaction in a microfluidic apparatus, the reaction is carried out using starting substances tagged with fluorophore and quencher. The detection of reaction products occurs according to the disclosure by a separation of fluorophore and quencher occurring in the context of the amplification reaction. For the detection reaction, at least one energy-transferring substance is added and the evaluation occurs on the basis of the fluorescence emission of the fluorophores which occurs.

Claims

1. A method for carrying out an amplification reaction in a microfluidic device, comprising: carrying out the amplification reaction using starting substances labeled with a fluorophore and a quencher; and detecting reaction products resulting from a separation of the fluorophore and quencher that has taken place in the course of the amplification reaction by adding at least one energy-transferring substance and evaluating the fluorescence emission of the fluorophores that occurs.

2. The method as claimed in claim 1, wherein the energy-transferring substance is a luminescent substance.

3. The method as claimed in claim 2, wherein the luminescent substance is 3-aminophthalhydrazide and/or 3-nitrophthalhydrazide.

4. The method as claimed in claim 1, wherein the detection is carried out in the presence of hydrogen peroxide.

5. The method as claimed in claim 4, wherein the hydrogen peroxide is used in the form of carbamide peroxide.

6. The method as claimed in claim 1, wherein the detection is performed in the presence of at least one catalyst.

7. The method as claimed in claim 6, wherein the catalyst is potassium hexacyanoferrate(III) and/or manganese peroxide and/or hydroquinone and/or catechol and/or resorcinol and/or horseradish peroxidase (HRP).

8. The method as claimed in claim 6, wherein the catalyst is initially introduced into the microfluidic device.

9. The method as claimed in claim 1, wherein the addition of the at least one energy-transferring substance includes adding the at least one energy-transferring substance by overcoating with a reaction liquid in which the at least one energy-transferring substance is contained.

10. The method as claimed in claim 1, wherein the fluorophore used is Rhodamine B, another Rhodamine, and/or Yakima Yellow and/or Cy5.

11. The method as claimed in claim 1, wherein the evaluation is performed includes using at least one optical filter.

12. The method as claimed in claim 1, wherein the amplification reaction is an endpoint reaction.

13. A kit for carrying out an amplification reaction in a microfluidic device, comprising: starting substances labeled with a fluorophore and a quencher; and at least one energy-transferring substance configured for a detection reaction.

14. The kit as claimed in claim 13, wherein: the starting substances labeled with a fluorophore and a quencher are configured for carrying out the amplification reaction; and the at least one energy-transferring substance is configured to detect reaction products resulting from a separation of the fluorophore and quencher that has taken place in the course of the amplification reaction and to enable evaluating the fluorescence emission of the fluorophores that occurs.

15. A microfluidic device for carrying out amplification reactions, comprising: starting substances labeled with a fluorophore and a quencher and configured to carry out the amplification reaction; and at least one energy transferring substance configured to detect reaction products resulting from a separation of the fluorophore and quencher that has taken place in the course of the amplification reaction and to enable evaluating the fluorescence emission of the fluorophores that occur.

Description

[0021] In the drawings:

[0022] FIG. 1 shows spectra for the absorption and emission of the Yakima Yellow fluorophore, and

[0023] FIG. 2 shows an emission spectrum of luminol.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0024] The proposed method provides a new method for carrying out an amplification reaction in a microfluidic device, in which method the detection method known per se for the amplification reaction based on fluorescent dyes is combined with chemical excitation, in particular non-radiative excitation of the fluorophores by transfer of energy from excited molecules. In the case of the fluorescence method that is known per se and used in connection with amplification reactions, starting substances labeled with a fluorophore, for example fluorophore-labeled primer chains or TaqMan probes, are incorporated or hybridized or hydrolyzed during the amplification reaction. These primers or probes contain one or more fluorophores and a quencher which are directly adjacent. For as long as this configuration of fluorophore and directly adjacent quencher exists, no fluorescence can be emitted. In particular, no fluorescence can be emitted from such primer chains or probes, in each case bearing a fluorophore and quencher, for as long as they are located freely in the solution. It is only the incorporation of such primer chains or probes into an amplification product that causes either the splitting and spatial separation of fluorophore and quencher or the cleavage of the quencher from the molecule bearing the fluorophore. Such fluorophores can then emit fluorescence radiation after excitation and represent a measure of the amount of amplification products formed. This method makes it possible, by way of the transfer of energy to the fluorophores on the basis of a chemical reaction, to dispense with complicated and expensive optics that were conventionally required for optical excitation in the context of the fluorescence detection reaction.

[0025] The proposed method can be carried out in particular for an endpoint PCR reaction or an isothermal endpoint amplification reaction in singleplex or multiplex in a silicon microarray or in some other microfluidic device, wherein the amplification reaction involves the described incorporation of molecule pairs consisting of quenchers and fluorophores and/or the hydrolysis of such starting substances during the formation of the amplification products. After completion of the amplification reaction, that is to say as soon as the amplification reaction in the array cells has essentially reached its endpoint, the microfluidic device can be overcoated with a reaction liquid containing the energy-transferring substances, in particular the luminescent substances. After they have been excited, said substances can carry chemical energy for a relatively long period of time (regardless of their luminescence), and so this energy can be transferred non-radiatively to the fluorophores.

[0026] The reaction on which the excitation of the energy-transferring substances is based, in particular the oxidation reaction, can be accelerated by a catalyst. Particularly suitable for this purpose are red prussiate (potassium hexacyanoferrate(III)) or hydroquinones or other oxidation catalysts. Said catalyst may be admixed with the reaction mixture consisting of hydrogen peroxide, luminol and suitable buffers. Some catalysts may also be prestored directly in the array cells of the microfluidic device provided that they do not interfere with the actual amplification reaction, this being the case for example for many hydroquinones. Other options for suitable catalysts include, for example, manganese peroxide (manganese dioxide) that may also be prestored 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. On contact with hydrogen peroxide, manganese peroxide catalyzes the decomposition of the hydrogen peroxide molecules and the oxidation of the energy-transferring substances, for example of luminol, to give the long-lasting excited form. Furthermore, manganese peroxide is a very cost-effective and safe compound that is therefore particularly suitable as a solid catalyst for the decomposition of hydrogen peroxide and the initiation of the subsequent oxidation of luminol.

[0027] Suitable fluorophores are in principle all fluorescent dyes that emit with a greater wavelength than the energy-transferring or luminescent substance used. By way of example, suitable fluorophores that may be used in combination with luminol as luminescent substance are Rhodamine B, other Rhodamines, Yakima Yellow or Cy5. FIG. 1 illustrates absorption and emission spectra of Yakima Yellow. Here the absorption maximum is 525 nm and the emission maximum is 550 nm and the emission extends to significantly longer wavelengths beyond 600 nm. FIG. 2 depicts the emission spectrum of luminol: the emission maximum is 425 nm and the emission extends to significantly greater wavelengths. The excitation energy of luminol can thus lead to excitation of Yakima Yellow. It is advantageous for the evaluation of the fluorescence emission of Yakima Yellow that the two emissions, i.e. the interfering luminescence emission of luminol and the fluorescence emission of Yakima Yellow, can easily be separated from one another by optical filters, so that the luminescence emission of luminol does not interfere with or overlay the fluorescence emission that is relevant for the evaluation of the detection reaction.

[0028] Customary probes with the Yakima Yellow fluorophore and a quencher are based in principle on a bridge structure that couples the quencher to the fluorophore. When a probe that has been labeled in this way is annealed or attached to a DNA strand, the bridge opens, as a result of which the quencher is spatially separated from the Yakima Yellow fluorophore.

[0029] The type of read-out in the sense of an endpoint PCR or an isothermal endpoint amplification reaction can be technically implemented without any great effort. Although the individual reaction does not allow any quantitative statement to be made, the “quantitative” feature is generally no longer necessary with the high degrees of multiplexing that are possible. The particular advantage of the proposed method, where optical excitation of the sample volumes is dispensed with, makes it possible for example to process and read even large-area silicon microarrays having a very large number of array cells and different detection reactions in parallel.

[0030] When carrying out the method, an amplification reaction that is known per se with primers and/or probes that are suitable for a fluorescence read-out can be carried out first. After the endpoint of the amplification reaction has been reached, the reaction mixture for the detection reaction according to the invention, for example consisting of water, luminol, hydrogen peroxide/urea (carbamide peroxide) and red prussiate (potassium hexacyanoferrate(III)) as catalyst for the reaction leading to the excitation of the energy-transferring substances, in the microfluidic device can be slowly fed to the PCR multiarray chip, so as to thereby slowly overcoat the reaction mixture of the amplification reaction. To assist the detection reaction, in particular to destabilize the hydrogen peroxide and to increase the solubility of luminol, it is for example also possible to add a small amount of sodium hydroxide solution or alkaline buffer (e.g. sodium hydrogencarbonate), it also being possible to contemplate prestorage of suitable buffers in the microfluidic device.

[0031] The substances for the reaction that serves for the excitation of the energy-transferring substances may be directly added at the start of the detection reaction or in some cases already prestored in the array cells or elsewhere in the lab-on-chip system. It is a particular advantage of such lap-on-chip systems that all the reagents required can be prestored therein at a suitable location and in a suitable manner. In particular, a PCR-compatible catalyst such as hydroquinone is suitable for prestorage in the array cells of the silicon chip as hydroquinone does not interfere with the amplification reaction. Another particularly suitable option is the prestorage of manganese peroxide (manganese dioxide) as water-insoluble solid catalyst in the array cells. Manganese peroxide, as a water-insoluble substance, also generally does not interfere with the amplification reaction that takes place in aqueous solution. Because manganese peroxide is a very efficient decomposition catalyst for hydrogen peroxide and hence for the oxidation of the luminol, manganese peroxide is also particularly suitable as a starting point for the chemical excitation of the luminescent substance, in particular of luminol. The excited luminol transfers its energy to the fluorophore, for example Yakima Yellow. Provided that the Yakima Yellow fluorophore is spatially separated from its quencher, that is to say in particular when the Yakima Yellow primer or the Yakima Yellow probe is bound to a DNA strand and the quencher is thereby removed, Yakima Yellow can fluoresce. For the read-out of the fluorescence radiation, use is preferably made of an optical filter (e.g. 570-580 nm), optionally combined with an additional edge filter that blocks emission with a wavelength shorter than 580 nm, in order to suppress the luminescence radiation of luminol that interferes with the evaluation.

[0032] Reaction mixtures used to carry out the PCR in the array cells are, for example, commercially available proprietary PCR kits from manufacturers which are themselves not subject matter of the present invention. The PCR kit selected is optionally supplemented by suitable PCR-compatible catalysts and additional buffer substances in order to optimally adjust said kit to the environment of the array cells in the silicon chip. Furthermore, the PCR kit should preferably work with Yakima Yellow or a suitable Rhodamine as the detection fluorophore in the sense of the present description.

[0033] An exemplary reaction mixture for the overcoating of the PCR kit after completion of the PCR in the array cells to trigger the detection reaction may comprise the following components and concentrations: [0034] Ultrapure water [0035] Hydrogen peroxide, 0.1%-10% (corresponding to 30 mmolar-3 molar), preferably 0.5%-5% (corresponding to 150 mmolar-1.5 molar) [0036] Alternatively carbamide peroxide (hydrogen peroxide-urea), 30 mmolar-3 molar, preferably 150 mmolar-1.5 molar [0037] Sodium hydroxide, 10 mmolar-1 molar, preferably 50-500 mmolar, particularly preferably 100-150 mmolar [0038] Alternatively sodium hydrogencarbonate, 50 mmolar-1 molar, preferably 100-500 mmolar [0039] Luminol, 10 mmolar 150 mmolar, particularly preferably 50-100 mmolar [0040] Catalyst (hydroquinone, horseradish peroxidase HRP, manganese peroxide, potassium hexacyanoferrate(III)), 10 mmolar-1 molar, preferably 50 mmolar-500 mmolar, particularly preferably 100 mmolar