Method for quantifying a nucleic acid solution and microfluidic analysis device

Abstract

The invention relates to a method for quantifying a first solution, containing at least one nucleic acid, comprising: thinning (32) the first solution with a second solution containing at least one intercalating fluorescent dye; carrying out (33) a fluorescence measurement on the first solution in order to obtain a fluorescence signal; carrying out a predefined number of repetitions of the two preceding steps; plotting the fluorescence signals against a thinning of the first solution; fitting (42) a function to the fluorescence signals; determining (43) a local maximum of the function or its first derivative; and determining (44) a concentration of the nucleic acid from a fluorescence signal, coming before the local maximum.

Description

[0001] Method for quantifying a nucleic acid solution and microfluidic analysis device

[0002] The present invention relates to a method for quantifying a solution containing at least one nucleic acid. Furthermore, the present invention relates to a microfluidic analysis device configured to perform the steps of the method.

[0003] State of the art

[0004] To quantify DNA using an intercalating fluorescent dye, a defined amount of the fluorescent dye, whose emission increases significantly upon intercalation or binding of nucleic acids, is added to the DNA solution to be measured, and the fluorescence intensity is then measured. Commonly used fluorescent dyes are ethidium bromide, propidium iodide, DAPI (4',6-diamidino-2-phenylindole), cyanine dyes, or cyanine-based dyes. The measured value is compared with a similarly measured standard, and the previously unknown amount of DNA is determined from this. To ensure that the measurement is performed in a concentration range in which there is a linear relationship between the DNA concentration and the fluorescence intensity, the maximum and minimum amount of DNA that can be detected using the method is defined beforehand.When measuring a sample with too high a concentration, an upper limit for the fluorescence intensity is established. If this limit is exceeded, the concentration can no longer be directly determined from the intensity measurement, as it falls outside the linear measurement range. EP 3 266 880 A1 describes a fluorescence-based quantification of a target nucleic acid using a data set from a quantitative amplification of reference samples and a data set from a quantitative amplification of the target nucleic acid. A reference table is generated from the data set of the reference samples, which is used to quantify the target nucleic acid. The data sets each describe a curve that is fitted with a sigmoidal function.

[0005] Disclosure of the invention

[0006] In the method for quantifying a first solution containing at least one nucleic acid, in particular DNA, the first solution is first diluted with a second solution containing at least one intercalating fluorescent dye. An intercalating fluorescent dye is understood in particular to be a fluorescent dye whose emission increases upon intercalation or binding of nucleic acids. Fluorescent dyes used can be, in particular, ethidium bromide, propidium iodide, DAPI (4',6-diamidino-2-phenylindole), cyanine dyes, or cyanine-based dyes. This not only reduces the concentration of nucleic acid in the first solution, but also brings it into contact with the fluorescent dye, allowing the dye to intercalate or bind with the nucleic acid.Mechanisms leading to such behavior can include: stiffening of the molecular structure due to binding to the nucleic acid scaffold, resulting in less vibrational relaxation and higher fluorescence quantum yields, and / or charge induction and stronger polarization due to coupling to nucleic acids (negatively charged nucleic bases), which induces higher dipole moments for optical transitions, resulting in stronger absorption and fluorescence (higher transition matrix elements). A fluorescence measurement is then performed on the first solution to obtain a fluorescence signal. The steps of dilution and performing the fluorescence measurement are repeated several times, with the number of repetitions being predetermined. After each dilution, a fluorescence measurement is performed on a less concentrated solution of the nucleic acid.Since the concentration of the nucleic acid is not yet known, the fluorescence signals obtained cannot yet be assigned to concentrations of the nucleic acid, but initially only to dilution levels.

[0007] After the specified number of dilutions and fluorescence measurements have been performed, the fluorescence signals are plotted against a dilution of the first solution. A first dilution level, a second dilution level, and so on are plotted on the abscissa, while the fluorescence intensities attributable to each dilution level are plotted on the ordinate. A function is then fitted to the fluorescence signals. This function can be a sigmoidal, polynomial, exponential / potential, or logarithmic function. A local maximum of the function or a local maximum of a first derivative of the function is determined. In particular, the local maximum closest to the highest dilution level must be selected. This is referred to below as the relevant local maximum.A nucleic acid concentration is then determined from a fluorescence signal that lies before the local maximum, in particular before the relevant local maximum. "Before" here means that the dilution level of the fluorescence signal used to determine the concentration is higher than the dilution level of the local maximum. This can be determined, in particular, by comparison with a similarly measured standard.

[0008] This method allows intrinsic control over which dilution level is optimal for quantification within the linear range of the relationship between nucleic acid concentration and fluorescence intensity. The occurrence of a local maximum, especially a relevant local maximum, of the function serves as an indicator to detect that the linear range has been exceeded. The method is therefore suitable for quantifying a solution containing a nucleic acid in an automated, particularly microfluidic, system where manual dilution and subsequent re-measurement of the solution is not possible.This goal could not be achieved more simply by setting a maximum fluorescence intensity above which the linear range is assumed to be left, since lower measured values ​​could occur due to interference from some fluorescent dyes, for example fluorophores.

[0009] The dilution and fluorescence measurement steps are preferably repeated at least four times each. This ensures that sufficient fluorescence signals are available to both fit the function and find a suitable measured value below the local maximum for concentration determination.

[0010] The concentration is preferably determined from a fluorescence signal that lies at least two dilution levels upstream of the local maximum, in particular upstream of the relevant local maximum. A further preferred determination of the concentration can also be carried out at the dilution level that is closest to the point with the highest positive slope upstream of the local maximum, in particular upstream of the relevant local maximum. When using the fluorescence signal that lies immediately upstream of the local maximum, in particular upstream of the relevant local maximum, there is a risk that this signal would already lie outside the range of a linear relationship between the concentration of the nucleic acid and the fluorescence intensity. A fluorescence signal lying even further upstream of the local maximum, on the other hand, would already exhibit an unnecessarily high dilution.

[0011] In a preferred embodiment of the method, the local maximum of the function, in particular the relevant local maximum, is determined from a first derivative of the function. In another preferred embodiment of the method, the local maximum of the first derivative of the function, in particular the relevant local maximum, is determined from the second derivative of the function.

[0012] If it turns out that the function and its first derivative do not exhibit a local maximum, especially a relevant local maximum, then all measured fluorescence signals lie within the range of a linear relationship between the nucleic acid concentration and the fluorescence intensity. In this case, it is preferable to determine the concentration from the fluorescence signal of the first fluorescence measurement, since the first solution is the least diluted in this measurement.

[0013] The method can be used to operate a microfluidic analysis device configured to perform the steps of the method. For this purpose, the microfluidic analysis device comprises, on the one hand, structural means for repeatedly performing the steps of dilution and performing the fluorescence measurement. On the other hand, it comprises means for plotting the fluorescence signals over the dilution of the first solution, fitting a function to the fluorescence signals, determining a local maximum of the function, and determining a concentration of the nucleic acid from the fluorescence signal that lies before the local maximum. For this purpose, these method steps are implemented, in particular, as a computer program.

[0014] Short description of the drawings

[0015] Embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description.

[0016] Figure 1 schematically shows part of a microfluidic analysis device according to an embodiment of the invention.

[0017] Figure 2 shows a flowchart of a method according to an embodiment of the invention.

[0018] Figure 3 shows a diagram showing a relationship between a DNA concentration and a fluorescence intensity in an embodiment of the invention.

[0019] Figure 4a shows a diagram of a polynomial function representing a relationship between DNA concentration and fluorescence intensity in one embodiment of the invention. Figure 4b shows a diagram of the first derivative of the polynomial function according to Figure 4a.

[0020] Embodiments of the invention

[0021] Figure 1 shows elements of a microfluidic analysis device 10 according to an embodiment of the invention. These elements are partially arranged in a fluidic layer of a microfluidic cartridge and partially arranged in an analysis device that accommodates the microfluidic cartridge. A first microfluidic channel 11 is arranged in the fluidic layer and opens into a first microfluidic chamber 12. It is designed to transport a first solution 21 into the first microfluidic chamber 12. The first solution 21 is an aqueous solution of DNA in a buffer medium. The concentration of the DNA is unknown. The first microfluidic chamber 12 is connected to a second microfluidic chamber 14 via a second microfluidic channel 13. A second solution 22 is stored in the second microfluidic chamber 14.The second solution 22 contains an intercalating fluorescent dye (for example, contained in the Qubit™ IX dsDNA HS Working Solution from Thermo Fisher Scientific). The first microfluidic chamber 12 has a transparent window, above which a fluorescence sensor 15 of the analyzer is arranged. The fluorescence sensor 15 has a light source to excite fluorescence in the first solution 21 and a sensor to detect the fluorescence response. It is connected to an electronic computing device 16, which is also connected to a pneumatic manifold (not shown) of the analyzer. A pneumatic layer of the cartridge can be controlled via the pneumatic manifold to control fluid flows in the fluidic layer.

[0022] Figure 2 illustrates the sequence of a method according to an embodiment of the invention. This is implemented as a computer program in the electronic control unit 16. After the start 30 of the method, the first solution 21 is initially introduced 31 into the first microfluidic chamber 12 by pumping it through the first microfluidic channel 11. Subsequently, the first solution 21 is diluted 32 with the second solution 22, with the dilution ratio in the present embodiment being 1:2. The dilution is performed by pumping a portion of the second solution 22 stored in the second microfluidic chamber 14 through a second microfluidic channel 13 into the first microfluidic chamber 12. Subsequently, a fluorescence measurement is performed 33 on the first solution 21 using the fluorescence sensor 15.Process steps 32 and 33 are repeated until a test 34 shows that six dilutions of the first solution have been carried out and six fluorescence signals have been recorded.

[0023] The fluorescence signals are now plotted 41 by diluting the first solution. Figure 3 shows a diagram illustrating the relationship between the concentration C of DNA in the first solution 21 and the measured fluorescence intensities I in relative units (RFU = relative fluorescence unit). It can be seen that a linear relationship between the concentration C and the fluorescence intensity I exists only for the three lowest concentrations. Since the concentration C is not yet known, the plot is instead plotted using dilution steps. The highest concentration C shown in Figure 3, 5 ng / l, corresponds to the first dilution step, and each subsequent halving of the concentration C corresponds to a further dilution step.

[0024] A polynomial function F is now fitted 42 to the fluorescence signals. The fitted function F is shown in Figure 4a. This is followed by determining 43 a local maximum M of the function F by calculating the first derivative F' of the function F. This is shown in Figure 4b. The position of the maximum M in the function F is shown in Figure 4a by means of a dashed line connecting Figure 4b with Figure 4a. Alternatively, a local maximum M' of the first derivative F' of the function F is calculated. This can be done by forming the second derivative.

[0025] A check 44 then follows to determine whether a local maximum M or M' has been found. If this is the case, as shown in Figures 4a and 4b, the concentration C is determined 45 from the fluorescence signal h that lies before the local maximum M or M'. The relationship between the fluorescence intensity I and the concentration C is established by a reference measurement. A chamber containing the reference sample is also arranged in the fluidic layer of the microfluidic cartridge, but is not shown in Figure 1.

[0026] If the test 44 shows that no local maximum M or M' was found, the concentration C is determined 46 from the fluorescence signal of the first execution 33 of the fluorescence measurement.

[0027] After the concentration C has been determined by one of the process steps 45 or 46, the process is terminated 47.

Claims

Claims 1. A method for quantifying a first solution (21) containing at least one nucleic acid, comprising the following steps: a) diluting (32) the first solution (21) with a second solution (22) containing at least one intercalating fluorescent dye, b) performing (33) a fluorescence measurement on the first solution (21) to obtain a fluorescence signal, c) performing a predetermined number of repetitions of steps a) and b), d) plotting (41) the fluorescence signals over a dilution of the first solution, e) fitting (42) a function (F) to the fluorescence signals, f) determining (43) a local maximum (M, M') of the function (F) or its first derivative (F'), and g) determining (45) a concentration (C) of the nucleic acid from a fluorescence signal that lies before the local maximum (M, M').

2. Method according to claim 1, characterized in that steps a) and b) are each repeated at least four times.

3. Method according to claim 1 or 2, characterized in that the determination (44) of the concentration takes place from a fluorescence signal (h) which is at least two dilution stages before the local maximum (M, M').

4. Method according to one of claims 1 to 3, characterized in that the local maximum (M) of the function (F) is determined from the first (F') derivative of the function (F).

5. Method according to one of claims 1 to 4, characterized in that the local maximum (M') of the first (F') derivative of the function (F) is determined from the second (F") derivative of the function (F).

6. Method according to one of claims 1 to 5, characterized in that the concentration (C) is determined (46) from a fluorescence signal from the first implementation of the fluorescence measurement if the function (F) and its first derivative (F') do not have a local maximum (M, M').

7. Microfluidic analysis device (10), characterized in that it is set up to carry out the steps of the method according to one of claims 1 to 6.

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