Chaotropic solution and use thereof for a microfluidic device

Abstract

The invention relates to a chaotropic solution, in particular for a microfluidic device, containing guanidinium thiocyanate and at least one disulfide bond-breaking compound, preferably mercapturic acid. The solution has a pH of less than 7.0, preferably 3.5 - 5. It can be used as a transport medium and / or as a lysis buffer and / or as a binding buffer for at least one biological sample. The invention further relates to a microfluidic device (10) in which the chaotropic solution is pre-stored.

Description

[0001] R. 416878

[0002] - 1 -

[0003] Description

[0004] title

[0005] Chaotropic solution and its use for a microfluidic device

[0006] The present invention relates to a chaotropic solution. Furthermore, the present invention relates to uses of the chaotropic solution, in particular for a microfluidic device. Finally, the present invention relates to a microfluidic device, in particular a microfluidic cartridge, in which the chaotropic solution is pre-positioned.

[0007] State of the art

[0008] Microfluidic devices can be used for automated molecular diagnostics, which identify pathogens based on their genetic characteristics. These devices often use disposable cartridges containing pre-filled reagents, for example, to perform a polymerase chain reaction (PCR) of nucleic acids. A patient sample is introduced into a sample inlet chamber of the cartridge via a sample transport medium. Some of these transport media contain guanidinium thiocyanate as a chaotropic reagent, which, at high concentrations, lyses and thus kills pathogens present in the patient sample, allowing for the safe transport of infection-critical patient samples.The cartridge often uses buffer solutions containing high concentrations of guanidinium thiocyanate to create a sufficiently chaotropic state in the mixture of the sample liquid and the buffer solution. This ensures both the lysis of any pathogens present and adequate binding of released nucleic acids in downstream filter frits. Therefore, these buffers are commonly referred to as lysis buffers or binding buffers. R. 416878.

[0009] - 2 -

[0010] EP 2 156888 A2 describes a method for cell analysis using different lysis buffers. One of the described chaotropic lysis buffers contains 3 mol / l guanidinium thiocyanate and 2% beta-mercaptoethanol. It has a pH of 7.5. Mercaptoethanol is a highly toxic substance that cannot be used in a lab-on-a-chip environment, i.e., in a microfluidic cartridge, for compatibility reasons (dissolving plastics) and because of its high toxicity. R. 416878

[0011] - 3 -

[0012] Disclosure of the invention

[0013] In a first aspect, a chaotropic solution, particularly aqueous, is provided, containing guanidinium thiocyanate and at least one disulfide-breaking compound. This solution has a pH value of less than 7.0.

[0014] A chaotropic solution is understood to be a solution in which ordered hydrogen bonds in the water are disrupted.

[0015] This solution composition is based on the understanding that chaotropic solutions containing guanidinium thiocyanate are unstable in aqueous solutions and gradually decompose through oxidation reactions, especially through exposure to light. Sulfur is released in this process.

[0016] As a result, aqueous guanidinium thiocyanate solutions always contain a sulfur background. This sulfur binds to proteins and nucleic acids (DNA and RNA) of a biological sample introduced into the chaotropic solution. These proteins can originate from the sample material itself, such as proteins from saliva or a mucosal swab. Alternatively, the chaotropic solution may contain a solid component, such as gelatin, if it is intended to serve as a transport medium. In the presence of sulfur, protein clusters can form through the formation of disulfide bridges between protein fibers. These protein clusters have a slimy consistency and can block the inner surface of a filter frit in a microfluidic device, particularly a microfluidic cartridge, rendering it ineffective for nucleic acid binding.This results in a loss of genetic material from the biological sample for the subsequent detection reaction and thus a loss of detection sensitivity. In extreme cases, mucus plugs can even completely clog a filter frit and, due to filter failure, lead to a total failure of the detection reaction. R. 416878.

[0017] - 4 -

[0018] Another problem with sulfur binding to proteins and disulfide bond formation arises from the fact that proteins are also generally likely to be temporarily bound to the filter frit and subsequently eluted into the subsequent detection reaction, thus carrying over bound sulfur into the mostly PCR-based detection reaction. A certain degree of protein carryover, and consequently the carryover of sulfur bound to these proteins, cannot be prevented even by increased washing of the filter frit with wash buffer. Sulfur interferes with many PCR reactions, sometimes even causing the reaction to fail completely. Therefore, the introduction of sulfur in free or bound form must be strictly avoided in the interest of reliable detection reactions.

[0019] It is therefore planned to use a chaotropic solution containing at least one disulfide-breaking compound in addition to guanidinium thiocyanate. Furthermore, the chaotropic solution should have a pH value of less than 7.0. A disulfide-breaking substance does not significantly affect the stability of the guanidinium thiocyanate. It cannot prevent the decomposition of guanidinium thiocyanate, particularly under the influence of light, but it can neutralize the harmful effects of the sulfur released in this process. The disulfide-breaking compound ensures that sulfur cannot form sulfur clusters or sulfur nanoparticles through bridging between sulfur atoms, or sulfur-protein conglomerates through bridging with proteins or nucleic acids, which would bind to a filter frit and subsequently be carried over into the detection reaction.It further prevents sulfur atoms, sulfur clusters, or sulfur nanoparticles from binding to proteins that are attached to the filter frit and subsequently carrying the bound sulfur into the detection reactions. It also prevents sulfur from agglomerating proteins into slimy clusters that could cover and disable binding sites on the filter frit, thereby blocking the retention of the sample's genetic material. This would result in a loss of genetic sample material and, consequently, a reduction in detection sensitivity.

[0020] In principle, disulfide-breaking substances also suffer from oxidation reactions, which affects their stability and their disulfide-breaking properties. R. 416878

[0021] - 5 -

[0022] properties would degrade over time. However, the presence of guanidinium thiocyanate, especially in high molar concentrations, which acts as an antioxidant, protects the added disulfide-breaking substance from oxidation and thus stabilizes it over a long period. A low pH of the solution also improves the binding behavior of genetic material such as DNA and / or RNA to a filter frit, so this low pH of the solution should be aimed for by appropriately selecting the disulfide-breaking substance.

[0023] Preferably, the pH of the solution is in the range of 1.5 to 5.5. A lower pH could lead to such a high concentration of thiocyanate ions reacting to form thiocyanic acid that it would no longer be stable in the solution, resulting in decomposition to hydrogen cyanide and perthiocyanic acid. However, the outgassing of hydrogen cyanide from the chaotropic solution would be unacceptable from a safety perspective. In particular, the pH of the solution is ensured to remain within the preferred range by appropriate selection of the disulfide bond-breaking agent and / or by the addition of suitable buffers (for example, calcium or magnesium phosphates).

[0024] The solution preferably has a pH value in the range of 3.5 to 5.5. Most preferably, the pH value is in the range of 3.5 to 5.0. This pH range is particularly advantageous for certain detection reactions to be carried out with a biological sample. To adjust the pH value within this range, the chaotropic solution may contain, in particular, Tris-HCl, sodium hydroxide, sodium hydrogen phosphate, or sodium bicarbonate.

[0025] The disulfide-breaking compound is preferably a mercapto compound. Particularly preferred are mercapturic acid or a mercaptoacetate, such as sodium thioglycolate or ammonium thioglycolate. Mercapturic acid is especially preferred because, in addition to its disulfide-breaking properties, it allows the chaotropic solution to be adjusted to the desired low pH between 3.5 and 5.5, depending on the concentration of mercapturic acid. R. 416878

[0026] - 6 -

[0027] The concentration of the disulfide-bridging compound in the chaotropic solution is preferably in the range of 0.005 wt% to 5.000 wt%. It is particularly preferably in the range of 0.100 wt% to 5.000 wt%. While the lower limit of this concentration range has proven advantageous for providing the chaotropic solution with sufficient long-term disulfide-bridging properties to compensate for adverse effects from sulfur released due to the oxidation of guanidinium thiocyanate, the upper limit is advantageous for preventing the formation of hydrogen cyanide. However, an acidic pH is generally desirable because it offers very advantageous assay properties, particularly during the binding process in the filter frit. Even if thiocyanate anions form thiocyanic acid in an acidic environment, it remains stable in aqueous solution up to a concentration of 5 wt%.Only at higher thiocyanic acid concentrations is there a risk that free hydrogen cyanide will form as a result of decomposition.

[0028] Guanidinium thiocyanate combines the excellent chaotropic properties of the guanidinium cation, as described by the Hofmeister series, with those of the thiocyanate anion. The concentration of guanidinium thiocyanate in the chaotropic solution is preferably in the range of 5 mol / L to 10 mol / L. This high concentration is also sufficient to reverse the antichaotropic behavior of other components of the solution, which may be added by the manufacturer to enable its use as a transport medium, and to transform it into an overall chaotropic state. Furthermore, such a high concentration of guanidinium thiocyanate protects a disulfide-breaking substance present in lower concentrations in the chaotropic solution from oxidation and thus stabilizes it over a long period.

[0029] The chaotropic solution can be used, in particular, as a transport medium and / or as a lysis buffer and / or as a binding buffer, or as part of one of these buffers, for at least one biological sample. In particular, R. 416878

[0030] - 7 -

[0031] Use as a lysis buffer and / or binding buffer is preferably carried out in a microfluidic device.

[0032] In a further aspect, a microfluidic device, in particular a microfluidic cartridge, is provided in which the chaotropic solution according to the first aspect is pre-positioned, particularly in a chamber of the device or cartridge. When a biological sample, optionally in a transport medium, is introduced into the microfluidic device for processing and analysis, it can be mixed with the chaotropic solution within the microfluidic device. Furthermore, the microfluidic device includes, in particular, at least one filter frit.

[0033] In a process step on the microfluidic device, the disulfide-breaking substance can pass through the filter frit unimpeded during a binding step and thus enter a waste container of the microfluidic device directly. Residues in the filter frit are washed out towards the waste container during washing steps, so that the eluate for the usually PCR-based detection reaction is pure and essentially free of disulfide-breaking substances from the chaotropic solution. This ensures that the disulfide-breaking substance does not enter the actual detection reaction and cannot have a detrimental effect on it.However, a positive influence on the detection reaction arises from the fact that in the acidic pH range of the chaotropic solution, the filter frit has particularly good binding properties for the genetic material of the sample, thus achieving a high detection rate and little carryover of sulfur into the detection reaction.

[0034] Brief description of the drawing

[0035] An embodiment of the invention is shown in the drawing and is explained in more detail in the following description. R. 416878

[0036] - 8 -

[0037] Figure 1 shows a partially cut-away side view of a microfluidic cartridge as a microfluidic device according to an embodiment of the invention.

[0038] Exemplary embodiments of the invention

[0039] In a first embodiment of the invention, this relates to a chaotropic solution intended for use as a transport medium for a biological sample. This chaotropic solution contains, for example, 5 mol / l of guanidinium thiocyanate and has a pH of approximately 7. A disulfide-breaking substance, for example mercapturic acid, can be added to this solution, for example at a weight fraction of 3%, to adjust, for example, an acidic pH of 4.

[0040] In a second embodiment, the invention relates to a chaotropic solution intended for use at least as part of a lysis buffer and / or binding buffer. This solution has the following composition: for example, 5 mol / l guanidinium thiocyanate and, for example, 3 wt% mercapturic acid to, for example, adjust to an acidic pH of 4.

[0041] In a third embodiment, the invention relates to a microfluidic cartridge 10. This is a disposable cartridge, such as those intended for use in the Vivalytic microfluidic analysis device (Robert Bosch GmbH, Germany). This cartridge has a fluidic carrier plate 11 with microfluidic channels. It also has a pneumatic carrier plate 12 with microfluidic channels. An elastomeric membrane 13 is arranged between the two carrier plates 11 and 12. By applying positive or negative pressure to the microfluidic channels of the pneumatic carrier plate 13, the elastomeric membrane 12 can be deflected. In this way, liquids can be transported in the microfluidic channels of the fluidic carrier plate 11.In a reagent chamber 20 in the fluidic support plate 11 of the cartridge 10, which is provided for receiving or pre-storing a lysis and binding buffer, the chaotropic R. 416878.

[0042] - 9 -

[0043] The solution according to the second embodiment is placed upstream. It can be transported through the microfluidic channels of the fluidic carrier plate 11 in order to bring it into contact with a biological sample introduced into the cartridge 10.

Claims

R. 416878 - 10 - Claims 1. Chaotropic solution, in particular for a microfluidic device (10), containing guanidinium thiocyanate and at least one disulfide bridge-breaking compound, characterized in that the solution has a pH value of less than 7.

0.

2. Chaotropic solution according to claim 1, characterized in that the solution has a pH value in the range of 1.5 to 5.

5.

3. Chaotropic solution according to claim 2, characterized in that the solution has a pH value in the range of 3.5 to 5.

0.

4. Chaotropic solution according to one of claims 1 to 3, characterized in that the disulfide bridge-breaking compound is a mercapto compound.

5. Chaotropic solution according to claim 4, characterized in that the disulfide-bridge-breaking compound is mercapturic acid or a mercaptoacetate.

6. Chaotropic solution according to any one of claims 1 to 5, characterized in that the concentration of the disulfide bridge-breaking compound in the chaotropic solution is in the range of 0.005 wt.% to 5.000 wt.%.

7. Chaotropic solution according to claim 6, characterized in that the concentration of the disulfide bridge-breaking compound in the chaotropic solution is in the range of 0.100 wt.% to 5.000 wt.%.

8. Chaotropic solution according to any one of claims 1 to 7, characterized in that the concentration of guanidinium thiocyanate in the chaotropic solution is in the range of 5 mol / l to 10 mol / l. R. 416878 - 11 - 9. Use of a chaoptropic solution according to any one of claims 1 to 8 as at least part of a transport medium and / or lysis buffer and / or binding buffer for at least one biological sample.

10. Use according to claim 9, characterized in that it is carried out in a microfluidic device.

11. Microfluidic device (10), in particular microfluidic cartridge (10) in which a chaotropic solution according to one of claims 1 to 8 is pre-positioned.

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