EP2189219A1 - Contamination barrier - Google Patents

Abstract

A contamination barrier contains at least one water-insoluble hydrocarbon compound or hydrocarbon mixture. An independent claim is also included for a process for avoiding contamination when processing aqueous solutions in open and/or automatic systems.

Description

The present invention relates to a contamination barrier and a method for avoiding contamination of aqueous solutions, which arise in particular by carry-over and / or aerosol formation, for example when pipetting on open systems.

In the field of biotechnology in particular, the search for automation solutions for safe and efficient processing, in particular for a large number of samples, such as those arising in the case of HIV virus monotoring, has been increasingly sought in recent years. Preferably, more and more fully automatic high-throughput workstations are required here, which process a very high number of samples in a very short time, and moreover an additional manual use is dispensed with, but at the same time even the smallest amounts of biological material are to be detected.

However, current robot systems have a fundamental problem when working with biological material, especially in the field of molecular biology and / or diagnostics. Since the samples are usually worked up in a plate-shaped body (such as a rack), the individual sample containers are open next to each other, it comes, mainly by aerosol formation, but also repeatedly by mechanical carryover of sample material, during processing to contamination of predominantly adjacent samples ,

To avoid such (cross) contamination, some additional modules and / or consumables for robot systems are already known in the prior art. For example, hoods or, as in the Gebrausmusterschrift DE 200 06 546 U1 illustrated covering mats for protecting a plurality of formed in a plate-shaped body and open up reaction tubes disclosed. However, this type of cover protects only the samples of a rack, for example, in the transport and / or storage condition. However, contamination of the individual samples on the rack continues to occur.

Further, the use of sterile consumables known in the art, such as specially coated disposable pipettes and / or disposable filter tips, may also be used the cross-contamination rate is reduced only, but not completely eliminated. Thus, contaminations, especially in the ppm range, continue to be a fundamental problem (for example in the field of PCR diagnostics, where single molecules are detected).

Furthermore, methods are known in which, as for example in the European patent EP 0011327 B1 represented, are added to the aqueous solution of water-soluble compounds to prevent aerosol formation. However, since they mix with the sample liquid and possibly even change it or lead to undesirable side reactions, their use is extremely disadvantageous and, in particular, not conceivable in PCR diagnostics.

Furthermore, to protect against contamination of the samples a variety of closure options, such as different covers, septa and / or filters, etc. were developed for single or multiple sample vessels. However, these have disadvantages in handling, especially in the field of automation, since, for example, the opening and closing of caps, etc., in particular individual samples, very time-consuming and depending on the closure system is hardly feasible by machine. In addition, there is still the risk that the aerosols produced in the sample vessel escape during processing and, in the subsequent step, when the adjacent sample container is opened, the sample can be contaminated with sample material.

In addition, sample containers with septa and / or thin filter materials can not be used variably in any robotic or automatic pipetting system, since only a few pipette tips and / or pipette needles are suitable for the use of such sample containers.

Since the state of the art does not disclose any known devices and / or a known method which could be used satisfactorily to avoid contamination, in particular in the field of automated PCR diagnostics, such a device or such a method is also disclosed can not derive from this, the present invention has the object to enable an efficient and reproducible processing of a high number of samples while avoiding contamination in the solutions to be analyzed in open and / or automated systems.

This object is achieved by providing a contamination barrier to avoid contamination of aqueous solutions in open and / or automated systems having at least one water-immiscible hydrocarbon and / or a hydrocarbon mixture. Thus, the object of the present invention is achieved by providing a method for preventing contamination in that, for processing aqueous solutions in open and / or automated systems, they are overcoated with at least one water-immiscible hydrocarbon or a hydrocarbon mixture.

The contamination barrier according to the invention is characterized in particular by its flexible use in a wide variety of sample vessels. It is particularly advantageous that the contamination barrier according to the invention can be easily and quickly formed in very small sample vessels and removed again. In particular, can be dispensed with an increased time-consuming and costly, manual or even mechanical use of caps and / or septa. A further advantage of the contamination barrier according to the invention is its flexible introduction into the sample vessel, whereby, in contrast to the use of septa and / or filter materials which are fixedly arranged in the sample vessel, the volume of the aqueous solution can be varied at any time without forming a cavity, in which there is a contamination-induced aerosol formation. Very particularly advantageous is the use in very small volumes of the aqueous solutions (eg in the ppm range and smaller).

Furthermore, for the coating of the contamination barrier according to the invention due to the water-immiscible hydrocarbons or hydrocarbon mixtures is an advantageous application to almost any aqueous solutions.

In order to avoid contamination when processing aqueous solutions in open and / or automated systems, at least one water-immiscible hydrocarbon is applied to the solution to be analyzed by overcoating. The contamination barrier according to the invention thus applied to the aqueous solution, like a film, lies completely on the aqueous solution and thus prevents the penetration as well as the formation of aqueous aerosols. Advantageously, the contamination barrier according to the invention preferably has at least one substituted or unsubstituted, branched or unbranched hydrocarbon. Furthermore, the inventive Contaminationsbarriere of a cyclic, saturated or unsaturated hydrocarbon (such as cyclohexane, etc.) or of an aromatic hydrocarbon (such as benzene or toluene, etc.) consist or are formed, but only those hydrocarbons are used, the not miscible with water. In addition, the hydrocarbons used according to the invention may carry as substituents, for example, one or more halogen atom (s), nitro group (s) and / or amino group (s).

All of the abovementioned (hydrocarbon) compounds can be present individually or else in mixtures (for example a hydrocarbon mixture such as mineral oil).

For the purposes of the present invention, mineral oil is understood as meaning the liquid distillation products obtained from mineral raw materials, such as, for example, crude oil, lignite and hard coal, wood or peat, which consist essentially of mixtures of long-chain, aliphatic and saturated hydrocarbons.
Particularly suitable are distillation products or hydrocarbon mixtures, such as white oil and / or other paraffin oils containing mainly long-chain alkanes, preferably having 13 to 20, particularly preferably 14 to 16 carbon atoms.

For the purposes of the invention, hydrocarbons are understood as meaning primarily branched or unbranched hydrocarbons which have 5 to 20, preferably 6 to 16, particularly preferably 8 to 12, carbon atoms. Very particular preference is given to using branched or unbranched alkanes having 8 to 12 carbon atoms, of which in turn octane, nonane, decane and / or dodecane and mixtures thereof are particularly preferred.

According to an alternative embodiment of the present invention, one or more water-immiscible additive (s) may be admixed to the contamination barrier of the invention. In the context of the invention, additives are understood as meaning hydrophobic substances which additionally contribute to the reduction or to the prevention of aerosol formation. Particularly preferred here is the addition of silicone oils in a wide variety of compositions and viscosities. An important property of silicone oils in this context is their inert behavior over others Substrates. Characteristic is also their high spreading capacity, with the formation of special properties, such as hydrophobicity, goes along.

For the purposes of the invention, silicone oils are understood primarily to mean synthesis oils based on semi-organic polymers and copolymers of silicon-oxygen units with organic side chains. Here, these unbranched chains constructed alternately of silicon and oxygen atoms preferably have a chain length of from 10 to 1000 silicon atoms, particularly preferably from 30 to 500 silicon atoms, very particularly preferably from 50 to 150 silicon atoms.

According to an advantageous embodiment of the present invention, the contamination barrier according to the invention (with and without additives) is preferably used for coating aqueous solutions in open and / or automated systems with biological sample material. For the purposes of the present invention, biological sample material is understood as meaning biopolymers which may be naturally occurring macromolecules, such as, for example, nucleic acids, proteins or polysaccharides, but also synthetically produced polymers, as long as they contain the same or similar components as the natural macromolecules ,

Surprisingly, it has been found that an overlay of aqueous solutions, which preferably contain biological sample material, with hydrocarbons of the aforementioned type, in particular in the form of the contamination barrier according to the invention, especially in open, automated systems, results in efficient and reproducible avoidance of, above all, aerosol-related contaminations.

Hereinafter, the present invention will now be explained in more detail with reference to accompanying drawings and exemplary embodiments.

Fig. 1

die Bildung von Aerosolen aus einer wässrigen Lösung bei im Stand der Technik bekannten offenen Probengefäßen;

Fig. 2

eine auf eine wässrige Lösung überschichtete erfindungsgemäße Kontaminationsbarriere in einem offenen Probengefäß;

Fig. 3

einen schematischen aus dem Stand der Technik bekannten Pipetiervorgang aus einem offenen Probengefäß, bei dem ein Teil einer wässrigen Lösung unter Aerosolbildung entnommen wird;

Fig. 4

einen schematischen Pipetiervorgang aus einem offenen Probengefäß, bei dem ein Teil einer wässrigen Lösung unterhalb einer erfindungsgemäßen Kontaminationsbarriere ohne Aerosolbildung entnommen wird;

Fig. 5

einen schematischen Pipetiervorgang aus einem offenen Probengefäß, bei dem einer wässrigen Lösung unterhalb einer erfindungsgemäßen Kontaminationsbarriere ohne Aerosolbildung ein weiterer wasserlöslicher Teil hinzugegeben wird.

Fig. 6

Darstellung der Calls auf dem Array

It shows:

Fig. 1

the formation of aerosols from an aqueous solution in open sample vessels known in the art;

Fig. 2

a contamination barrier superimposed on an aqueous solution according to the invention in an open sample vessel;

Fig. 3

a schematic pipetting process known from the prior art from an open sample vessel, wherein a portion of an aqueous solution is taken under aerosol formation;

Fig. 4

a schematic Pipetiervorgang from an open sample vessel, in which a portion of an aqueous solution is removed below a contamination barrier according to the invention without aerosol formation;

Fig. 5

a schematic Pipetiervorgang from an open sample vessel, in which an aqueous solution below a contamination barrier according to the invention without aerosol formation, a further water-soluble part is added.

Fig. 6

Representation of the calls on the array

The in Fig. 1 and Fig. 3 It can be seen clearly from the prior art that mechanical loading during pipetting and / or mixing of the sample etc., for example by means of a pipetting device 6 or the like, in a commercially available reaction vessel 1 on a pipetting plate 2 (such as a microtiter plate, etc .) on a biological sample having aqueous solution 3, the aerosols 4 form.
Such aerosol formation can, as soon as the aerosol 4 flows out of the reaction vessel 1, easily lead to contamination of adjacent reaction vessels.

By in the Fig. 2 . 4 and 5 represented contamination barrier according to the invention 5 such a contamination-related aerosol formation is avoided because the aqueous aerosols 4 can not penetrate the contamination barrier 5. The water-insoluble contamination barrier 5 lies in the reaction vessel 1 like a film on the aqueous solution 3 in which the biological sample material is in dissolved form.

During the pipetting process, as in 4 and 5 shown in the reaction vessel 1 located aqueous solution 3 or the sample respectively processed under the contamination barrier 5. The water-insoluble composition of the contamination barrier 5 precludes the formation of aerosols 4 both during the immersion and retraction of the pipetting device 6 from the aqueous solution 3 and during mixing.

Also when in Fig. 5 As shown, adding another aqueous solution to the sample does not result in aerosol formation of the aqueous mixture 7 when mixing the aqueous solution 3 with the additional solution due to the contamination barrier 5 according to the invention.

Since the contamination barrier 5, in contrast to filters and / or septa firmly inserted into reaction vessels, is also very flexible, variable amounts of aqueous solution 3 can be removed or added to the reaction vessel 1 at any time by means of a pipetting device 6 without interposing between the surface of the reaction vessel 1 aqueous solution 3 and the contamination barrier 5 forms a cavity, which in addition the formation of aerosols 4 is avoided.

LIST OF REFERENCE NUMBERS

1

open reaction vessel

2

Pipetierplatte

3

aqueous solution

4

aerosol molecules

5

contamination barrier

6

pipetting

7

aqueous mixture (from sample solution added and added)

embodiments

On the basis of the following test procedures, the main sources of contamination (KQ) of open, automated pipetting systems, in particular in fully automated high-throughput workstations, so-called robotic systems (such as BioRobot Mdx / QIAGEN GmbH), and contaminations (K), mainly by aerosol formation , but also repeatedly caused by mechanical carryover of sample material, demonstrated and shown the significant reduction of such contamination by the use of the contamination barrier according to the invention.

As proof, cross-contamination tests were performed on samples with biological sample material in the ppm range and smaller, as they are used in common procedures in the field of PCR diagnostics. The experiments were based on one common methods known in the art for the isolation and purification of nucleic acids, in particular of viral RNA (protocol used: Q1Aamp 96 Virus Mdx V1.1 / QIAGEN GmbH).

Commercially available kits (eg QIAamp 96 Virus BioRobot Kit / QIAGEN GmbH) etc., which are particularly suitable for use in robotic systems, were also used as system accessories, reagents, consumables etc.
For carrying out the experiment with alkane template (Examples 1b and 2b), 100 μl of dodecane were manually introduced into common 96-well deepwell plates (hereinafter referred to as S-blocks).

The detection was not only by mere observations during the individual pipetting steps in the purification process, but also by the additional determination of actual nucleic acid contaminants in the negative samples in a subsequent downstream analysis (eg PCR, RT-PCR, etc.). In addition to the main sources of contamination of such systems, the examples below clearly show the effectiveness of the contamination barrier according to the invention on the basis of the determined contamination rates (KR).

Example 1:

1. a) mit einem Probenvolumen von 285 µl ohne Alkanvorlage und
2. b) mit einem Probenvolumen von 285 µl mit Alkanvorlage.

Cross-contamination tests performed

1. a) with a sample volume of 285 μl without alkane template and
2. b) with a sample volume of 285 μl with alkane template.

The automated process was started with the identification of the samples, the so-called loadcheck. As positive sample material (PP) hepatitis C virus material (arHCV) with an average concentration of 10 ⁸ - 10 ⁹ RNA copies / ml and as negative sample material (NP) negative plasma (eg citrated plasma / Breitscheid) was used. After the load check, 40 μl each of a commercially available protease (eg QIAGEN protease / QIAGEN GmbH) were pipetted into the S block and the system was heated to 56 ° C.

The addition of 285 .mu.l of sample material took place, as shown schematically below, in the "checkerboard pattern". In addition to each PP (+), an NP (-) was alternately pipetted into the S block (KQ 1!). <b> Table 1: </ b> Schematic representation of the plots of PP and NP in checkerboard pattern / S-block 1 2 3 4 5 6 7 8th 9 10 11 12 A + - + - + - + - + - + - B - + - + - + - + - + - + C + - + - + - + - + - + - D - + - + - + - + - + - + e + - + - + - + - + - + - F - + - + - + - + - + - + G + - + - + - + - + - + - H - + - + - + - + - + - +

Subsequently, 305 μl of a commercially available lysis buffer (eg Lysis Buffer AL / QIAGEN GmbH) were added to the individual reaction solutions, these were then mixed by pipetting up and down (KQ 2!) And subsequently incubated for 10 minutes. Finally, at intervals of about one minute per row of each reaction solution, 360 μl of ethanol (p.a. or min. 96%) were pipetted in each case twice (KQ 3!).

For the separation or purification of the nucleic acids isolated in this way, 910 μl of lysate were in each case automatically transferred to appropriate filter systems (eg QIAamp 96 plate / QIAGEN GmbH) (KQ 4!) And filtered under vacuum for 5 minutes at 25 ° C. (KQ 5! ).

According to the experimental protocol (see above), the filters loaded with the nucleic acid were subsequently under vacuum with in each case 800 μl of a commercially available washing buffer (eg Wash Buffer AW2 / QIAGEN GmbH) (KQ 6!) And then in a second washing step with 930 μl ethanol (pa or at least 96%) (KQ 7!) and then dried the membranes to remove ethanol under vacuum and 60 ° C in an automated vacuum device (eg RoboVac / QIAGEN GmbH) (KQ 8!).

The purified nucleic acid was finally eluted under vacuum for one minute with 50 μl and once with 100 μl of at least one commercially available elution buffer (eg Elution Buffer AVE / QIAGEN GmbH) from the filter system (KQ 9!). The collected eluate was processed outside the automated pipetting system for the subsequent amplification process. To the eluate prepared for the RT-PCR, a commercially available enzyme mix (eg Mastermix / QIAGEN GmbH) was pipetted (KQ 10!), An RT-PCR was carried out to determine the nucleic acid contamination and the results were evaluated.

The two experimental runs (runs 1aI and 1aII) without introduction of dodecane were carried out with the following ar HCV dilution: $$1\times {10}^{11}\mathrm{IU}/\mathrm{ml}+148.5\mathrm{ml\; of\; NP}=1\times {10}^9\mathrm{IU}/\mathrm{ml}$$

Up to the addition of the elution buffer no observations could be made neither in the course 1aI nor in the course 1aII, which would have pointed to contamination in particular at the sources of contamination (KQ) 1-4.
Only in the course of the elution (KQ 7) could observations be made in this regard. Thus, for example, a distinct blistering was observed in the addition of the elution buffer, especially in row 12. Partially the bubbles burst when immersing and pulling out the pipette tips. After elution, the outlet openings of the filter system (Nozzles) were as yellow as those of the PP in row 1 at NP positions B, D, F (as well as in barrel 1a).

• Lauf 1aI: 9 K /48 NP ⇒ KR_{Lauf 1aI} = 18,75 %
• Lauf 1aII: 2 K /48 NP ⇒ KR_{Lauf 1aII} = 4,17 %

After carrying out the RT-PCR, the following cross-contamination was determined:

• Run 1aI: 9 K / 48 NP ⇒ KR _{Run 1aI} = 18.75%
• Run 1aII: 2 K / 48 NP ⇒ KR _{Run 1aII} = 4.17%

This results in an average contamination rate (CR) of 11.46%.

The three experimental runs (run 1bl, 1bll and 1blll) with the introduction of dodecane were also carried out with the abovementioned ar HCV dilution: $$1\times {10}^{11}\mathrm{IU}/\mathrm{ml}+148.5\mathrm{ml\; of\; NP}=1\times {10}^9\mathrm{IU}/\mathrm{ml}$$

Run 1bIII was followed after series 1b could not be evaluated in run 1bII due to the lack of PP task. However, during the course of the experiment, no particular incidents indicative of carryover of sample material during pipetting operations or aerosol formation could be observed.

• Lauf 1 bl: 1 K /42 NP ⇒ KR_{Lauf 1bI} = 2,38 %
• Lauf 1bII: 3 K /48 NP ⇒ KR_{Lauf 1bII} = 6,25 %
• Lauf 1bIII: 3 K/48 NP ⇒ KR_{Lauf 1bIII} = 6,25 %

After carrying out the RT-PCR, the following cross-contamination was determined:

• Run 1 bl: 1 K / 42 NP ⇒ KR _{Run 1bI} = 2.38%
• Run 1bII: 3 K / 48 NP ⇒ KR _{Run 1bII} = 6.25%
• Run 1bIII: 3 K / 48 NP ⇒ KR _{Run 1bIII} = 6.25%

This results in an average contamination rate (CR) of 4.96%.

After comparing the determined contamination rates it could be shown that by using dodecane, the cross-contamination rate for the entire isolation and separation process can be more than doubled (factor 2,3).

Example 2 :

1. a) mit einem Probenvolumen von 570 µl ohne Alkanvorlage und
2. b) mit einem Probenvolumen von 570 µl mit Alkanvorlage.

Cross-contamination tests performed

1. a) with a sample volume of 570 μl without alkane template and
2. b) with a sample volume of 570 μl with alkane template.

The automated process was started with the identification of the samples, the so-called loadcheck. As positive sample material (PP) hepatitis C virus material (arHCV) with an average concentration of 10 ⁸ - 10 ⁹ RNA copies / ml and as negative sample material (NP) negative plasma (citrated plasma / Breitscheid) was used. After the loadcheck each 80 μl of a commercially available protease (eg QIAGEN protease / QIAGEN GmbH) were pipetted into the S-block and the system was heated to 56 ° C.
The addition of each 570 .mu.l of sample material was carried out, as already schematically shown in Table 1, in the "checkerboard pattern". In addition to each PP (+), an NP (-) was alternately pipetted into the S block (KQ 1!).

Subsequently, 610 μl of a commercially available lysis buffer (eg Lysis Buffer AL / QIAGEN GmbH) were added to the individual reaction solutions, these were then mixed by pipetting up and down (KQ 2!) And subsequently incubated for 10 minutes. Finally, at intervals of one minute per row of each reaction solution, 720 μl of ethanol (p.a or min. 96%) were pipetted in each case twice (KQ 3!).

In order to separate or purify the nucleic acids isolated in this way, in each case two times 910 μl of lysate were transferred automatically into appropriate filter systems (eg QIAamp 96 plate / QIAGEN GmbH) (KQ 4!) And filtered under vacuum for 5 minutes at 25 ° C. (KQ 5 !).

According to the experimental protocol (see above), the filters loaded with the nucleic acid were subsequently under vacuum in each case with 800 μl of a commercially available washing buffer (eg Wash Buffer AW2 / QIAGEN GmbH) (KQ 6!) And then in a second washing step with 930 μl ethanol (pa or at least 96%) (KQ 7!) and then dried the membranes to remove ethanol under vacuum and 60 ° C in an automated vacuum device (eg RoboVac / QIAGEN GmbH) (KQ 8!).

The purified nucleic acid was finally eluted under vacuum for one minute with 50 μl and once with 100 μl of at least one elution buffer (eg Elution Buffer AVE / QIAGEN GmbH) from the filter system (KQ 9!). The collected eluate was processed outside the automated pipetting system for the subsequent amplification process. To the eluate prepared for the RT-PCR, a commercially available enzyme mix (eg Mastermix / QIAGEN GmbH) was pipetted (KQ 10!), An RT-PCR was carried out to determine the nucleic acid contamination and the results were evaluated.

The two experimental runs (Run 2aI and 2aII) without the introduction of dodecane were carried out with the following ar HCV dilution: $$15\mathrm{<figure-callout\; id="1"\; label="ml"\; filenames="imgf0003.png"\; state="\{\{state\}\}">ml</figure-callout>}1\times {10}^9\mathrm{IU}/\mathrm{ml}+22.5\mathrm{ml\; of\; NP}=4\times {10}^{\mathrm{8th}}\mathrm{IU}/\mathrm{ml}$$

As soon as the lysis buffer is added to the S block, the bubbles already pile up to the upper edge of the individual reaction vessels. In addition, the pipetting tips (tips) drove out of the reaction vessels so quickly that some of the resulting bubbles burst (especially in positions H5 and H10). During the second addition of the lysate, blisters, which were pulled up and / or burst when the tips were pulled out, were also produced during mixing in the S block.

Even with the addition of the elution buffer, it was possible to make observations regarding contamination both in the course of 2 l and in the course of 2 l. For example, significant bubbling was seen during and after the addition of the elution buffer, particularly in row G and at position H6. Partly the bubbles also burst here when dipping and / or pulling out the tips. After elution in row 1 at NP positions B, D, F and H the nozzles under the QIAplate were also yellow as in the PP.

• Lauf 2I: 31 K /48 NP ⇒ KR_{Lauf 2aI} = 64,58 %
• Lauf 2aII: 34 K/48 NP ⇒ KR_{Lauf 2aII} = 70,08 %

After carrying out the RT-PCR, the following cross-contamination was determined:

• Run 2I: 31 K / 48 NP ⇒ KR _{Run 2aI} = 64.58%
• Run 2aII: 34 K / 48 NP ⇒ KR _{Run 2aII} = 70.08%

This results in an average contamination rate (CR) of 67.33%.

At this point, it clearly shows that the pipetting steps are more susceptible to cross-contamination as the sample volume increases, since the S-block volume is fully utilized here.

The two experimental runs (runs 2bI and 2bII) with the introduction of dodecane were also carried out with the abovementioned ar HCV dilution: $$15\mathrm{<figure-callout\; id="1"\; label="ml"\; filenames="imgf0003.png"\; state="\{\{state\}\}">ml</figure-callout>}1\times {10}^9\mathrm{IU}/\mathrm{ml}+22.5\mathrm{ml\; of\; NP}=4\times {10}^{\mathrm{8th}}\mathrm{IU}/\mathrm{ml}$$

In order to better observe the effect of dodecane coating on the reduction or prevention of blistering, etc., the dodecane used was previously stained with Sudan black.

During the entire experiment, however, no special incidents with regard to contamination could be observed. Also, the Nozzles on the QIAplate had no residues or discoloration after elution as already mentioned under Example 1b).

• Lauf 2bl: 6 K /48 NP ⇒ KR_{Lauf 2bI} = 12,50 %
• Lauf 2bII:10 K /48 NP ⇒ KR_{Lauf 2bII} = 20,83 %

After carrying out the RT-PCR, the following cross-contamination was determined:

• Run 2bl: 6 K / 48 NP ⇒ KR _{Run 2bI} = 12.50%
• Run 2bII: 10 K / 48 NP ⇒ KR _{Run 2bII} = 20.83%

This results in an average contamination rate (CR) of 16.67%

After comparing the determined contamination rates, it could thus be shown that by using dodecane, the cross-contamination rate for the entire isolation and separation process can even be reduced by at least a factor of 4 with higher sample and reaction volumes.

As already mentioned above, however, such an overall method has a number of sources of error or contamination. In order to determine the effect of the contamination barrier according to the invention for a single pipetting step, individual samples (preferably NP) were taken in the course of these test series and analyzed by means of RT-PCR. Based on the observations made in Examples 1b and 2b that the formation of bubbles or aerosols was prevented by the dodecane coating, in particular in the first process steps (in the area of the contamination sources 1 to 4) by the immersion and extraction of the pipette tips were Samples taken and analyzed from these points of the overall procedure. None of these samples had nucleic acid contaminants. For individual Process steps can thus even be completely avoided by the use of dodecane (cross) contamination.

Further experiments showed that the use of dodecane in the pipetting steps of the downstream analyzes (such as the RT-PCR) can be successful in reducing (cross) contamination. For example, in further cross-contamination tests, the commercially available enzyme mixture (eg Mastermix / QIAGEN GmbH) necessary for an RT-PCR was added to a sample coated with dodecane. Here, too, the negative samples in the test series with the contamination barrier according to the invention had significantly less or no contamination.

However, overlaying with the contamination barrier according to the invention is not only suitable for any kind of pipetting processes. According to a further embodiment of the present invention, the contamination barrier according to the invention can also be used in other modules of sample processing in which (cross) contamination occurs. Thus, for example, the use of the contamination barrier according to the invention also advantageously prevents cross-contamination caused by aerosol formation, when overturning with washing buffers, by dispensing with a dispenser, by introducing stirring or mixing devices (such as stirring fish, stirring bars, mixing punches) or mixing flask etc.) as well as mechanical stirring and / or mixing processes occurs. (Wherein stirring is meant to be a circular motion and mixing to be an up and down motion.)

In addition, the contamination barrier used can also positively influence other applications. Thus, for example, the use of the contamination barrier according to the invention in array experiments as shown below, in addition to the avoidance of contaminations, also leads to a stabilization of such an application.

Example 3 :

Due to the special properties of the additives according to the invention, blends of mineral and silicone oils have been selected in addition to mineral oils for the overlay of reaction mixtures for the array experiments.

The contamination barrier according to the invention was used in the course of preparation of biotin-labeled cRNA for hybridization on microarrays (eg Human Genome U133B Array / Affymetrix, US), different volumes of the contamination barrier being added to the cRNA fragmentation approaches. As a contamination barrier different amounts of mineral oil or a mixture of mineral oil and silicone oil were used.

The synthesis of the hybridization samples was performed according to the manufacturer's protocol (Affymetrix). After synthesis of cRNA from 2 μg Hela S3 total RNA, 1 μl, 5 μl and 10 μl of the oils were added to various fragmentation mixtures. These fragmentation approaches were transferred to the hybridization approach. Hybridization and analysis of the GeneChip arrays was performed according to the manufacturer's instructions. Samples (K) were used as controls, which were also processed without contamination barrier according to the standard manufacturer protocol for sample preparation.

Subsequently, data was collected for this series of experiments (on the background of the arrays used), on detectable, statistically relevant signals (Present Calls) and on the undetectable signals on the array. The fluctuations of the measured values should be small and independent of the addition of the oils. The data obtained were shown in a column diagram in FIG. 6 shown.

The results show that uniform hybridization results are achieved by adding the contamination barrier according to the invention (with and without additive). The fluctuations of the calls are +/- 2%, which corresponds to the fluctuation for technical replicas given by the chip manufacturer.

Furthermore, since it is also advantageous and desirable to avoid cross-contamination in the automated preparation of samples for hybridization to microarrays, in further experiments the effect of the contamination according to the invention on an automated system (BioRobot 8000 / QIAGEN GmbH) in which carryover of parts of the contamination barrier during the processing steps or during sample preparation. For the automated preparation of RNA from cell culture cells, the RNeasy 96 chemistry (QIAGEN GmbH) was used. For this 5 μg total RNA from Hela cells were bound to the column matrix of the RNeasy 96 plates (n = 4). After three washes, the purified RNA was removed from the column Water elutes. To avoid cross-contamination during elution and to improve the yield, the sample was previously overcoated with mineral oil and a mixture of mineral oil and silicone oil. In order to check whether the additive alone is also suitable for this application, a test batch was only covered with silicone oil. Subsequently, the RNA yield was determined photometrically.

Also, the overlaying of the samples with a contamination barrier according to the invention, to which a silicone oil was added as an additive, resulted on an automated system in a higher yield than comparable experiments without the contamination barrier according to the invention. Surprisingly, the yield could be increased by up to 10% with the use of the additives according to the invention, which represents a further improvement of the contamination barrier according to the invention.

Further series of experiments also showed that, in addition to the abovementioned mineral oil / silicone oil mixtures, silicone oils alone or in the form of additive mixtures in hybridization mixtures can also act as a contamination barrier according to the invention. (Commercially available silicone oils, such as AK 35, AK 50 and AK 100 from Wacker Chemie GmbH) were tested.

Claims (12)

Contamination barrier to prevent contamination of aqueous solutions in open and / or automated systems, the contamination barrier
consists of at least one silicone oil, or
a mixture of at least one silicone oil with at least one water-immiscible hydrocarbon or with a hydrocarbon mixture. Contamination barrier according to claim 1, characterized in that it comprises an unsubstituted hydrocarbon. Contamination barrier according to claim 1, characterized in that it comprises a substituted hydrocarbon. Contamination barrier according to one of claims 1 to 3, characterized in that it comprises a cyclic, saturated or unsaturated hydrocarbon. Contamination barrier according to one of claims 1 to 3, characterized in that it comprises a branched and / or unbranched acyclic hydrocarbon. Contamination barrier according to one of claims 1 to 5, characterized in that the hydrocarbon has 5 to 20, preferably 6 to 16, particularly preferably 8 to 12 carbon atoms. Contamination barrier according to one of claims 1 to 6, characterized in that it has as a hydrocarbon particularly preferably a branched or unbranched alkane, preferably having 8 to 12 carbon atoms. Contamination barrier according to claim 7, characterized in that it has as alkane preferably octane, nonane, decane and / or dodecane and mixtures thereof. Contamination barrier according to one of claims 1 to 8, characterized in that it comprises mineral oil as a hydrocarbon mixture. Contamination barrier according to one of claims 1 to 9, characterized in that the silicone oil comprises a synthetic oil based on semi-organic polymers and copolymers of silicon-oxygen units with organic side chains. Contamination barrier according to one of claims 1 to 10, characterized in that the silicone oil unbranched alternately constructed of silicon and oxygen atoms chains having a chain length of 10 to 1000 silicon atoms, preferably from 30 to 500 silicon atoms, particularly preferably from 50 to 150 Silicon atoms, has. Process for the prevention of contamination, characterized in that when processing aqueous solutions in open and / or automated systems, they are overlaid with a contamination barrier according to at least one of the preceding claims.

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