CA2389656A1 - Microstructured pipettes as metering systems - Google Patents

Abstract

The invention relates to pipettes with structured surfaces. The surfaces of the pipettes which come into contact with a liquid have surface elevations with an average height of 50 nm to 10 .mu.m, an average distance of 50 nm to 10 .mu.m and surface energies of less than 19 mN/m.

Description

CREAVIS Gesellschaft fur - 1 - O.Z. 5496 ' Technologie and Innovation mbH
PATENTE ~ MARKEN
Microstructured pipettes as metering systems Defined amount of liquids are frequently taken up and distributed using pipettes or similar tools. With the aid of pipettes, defined amounts of liquid can be taken up from a storage container and transferred into a second receptacle. In practice, accurate and reproducible dispensing of the amount of liquid is carried out by means of disposable pipette tips.
In particular in combinatorial chemistry and biotechnology, methods which 1 o require a large number of pipetting steps are employed today. These pipetting steps are frequently carried out fully automatically by so-called pipetting robots, which work through a preset program. Such systems enable a very high sample throughput to be achieved. However, since increasingly a large number of different batches are being employed for screening with extremely small amounts, ever-smaller sample batches must be selected. This requires ever-higher pipetting speeds in ever-smaller systems. A problem which has proven extremely technically difficult is detachment of the pipetted liquid from the pipette tip if the amounts of liquid are very small. This step is circumvented in industry today by 2 o immersing the pipette tip in the reaction volume. The disadvantage of this method is that immersion of the pipette into the reaction volume is accompanied by contamination of the pipette tip. In processes which do not permit contamination, it is then necessary to change the pipette tip or to clean it by a complex procedure. These steps are very expensive and time-consuming. In practice, there is a move today toward pipetting a solution taken up in one portion in a number of reaction volumes of equal consistency. This creation of so-called copies has the advantage that virtually no contamination occurs. The disadvantage of this method is, however, the complex handling of the reaction plates.
The abovementioned problems do not occur if the drop detaches from the pipette tip automatically. In today's commercially available systems, this typically takes place with drops in the region of a few p1. This is not a - 2 - O.Z. 5496 problem in the case of reaction plates having 96 wells, since the reaction - volume is significantly greater than 1 ml. In systems having 348 or 1536 wells, the pipetted volumes become smaller and smaller, since the actual reaction volume here drops to 10 NI. In such batches, volumes of from 10 n1 to 1 NI are typically pipetted. In the case of these reaction plates, detachment of a drop is only possible by immersing the pipette tip into the reaction volume. This results in the above-described contamination of the pipette tip or of the reaction liquid.
1 o Processes by means of which very small drops can be applied to a surface are known from the area of adhesive technology and ink jet technology.
DE 28 19 440 describes a process in which liquid is conveyed to a delivery nozzle via a tube from a storage tank located above the delivery nozzle.
Drops formed at the opening are detached by a pulse of compressed gas.
This process can also be utilized to detach a drop of liquid from a pipette tip and offers the advantage that extremely small drops can be applied to a surface. Disadvantages of the process are the poor reproducibility of the drops and that the pressure pulse can also push liquid out of the wells.
2 o DE 19 74 2005 describes a process by means of which even volumes of less than 100 n1 can be forced out of a thin capillary. The volume forced out is dependent on the diameter of the capillary and the applied pressure pulse. The disadvantages of this process are firstly the very complex pressure technology and the poor reproducibility of the drop size.
DE 197 42 005 describes a refinement of this process. A process has also been disclosed in which the pressure is generated by a piezoelectric method. The overpressure tank here is replaced by a piezomodulator mounted on the capillary. The advantages of this method are the improved reproducibility of the pulses and thus of the drop size and the simple 3 o electrical control.
The liquid to be pipetted is frequently intended to be introduced into solutions already introduced into the cup of a well. Since both the cup and the introduced solution have a small volume, the use of pressure pulses for - 3 - O.Z. 5496 pipetting entails the risk of some of the reaction solution being pushed out of the cup. The precision of the pipetted volume, i.e. the drop size here, is furthermore highly dependent on the detachment of the drop from the pipette tip or on the drop formation per se.
Drop formation has hitherto only been described inadequately in physical terms. When a drop of a liquid contracts, the hydrodynamic equations which normally reliably describe the behavior of liquids fail. S. Nagel in Physical Review Letters, Volume 83, 1999, pages 1147 ff. describes drop 1 o formation more precisely. Just before a drop detaches, it hangs on an extremely thin liquid filament, which rapidly contracts. This contraction is caused by the surface tension, which tries to keep the surface area of the flowing liquid as small as possible. Just before the filament breaks, it loses the properties of a liquid. This is where the hydrodynamic equations finally fail. Nagel has succeeded in describing drop formation very precisely by means of a suitable visualization method. However, it is not possible to derive the prerequisites for rapid drop detachment, such as, for example, the geometrical shape of a pipette tip, from the publication by Nagel and that by J. Lister in Physical Review Letters, Volume 83, 1999, pages 2 0 1151 ff.
Modern pipettes, pipette tips or dispensers for taking-up or distributing liquids should satisfy the following conditions:
- suitable even for liquid amounts of less than 1 NI
2 5 - do not use pressure pulses - do not use antimicrobicidal materials - no "entrainment" of reaction media on immersion of, for example, pipette tips or capillary tips in liquids due to residues of these liquids.
3 o The object of the present invention was therefore to provide pipettes or pipette tips by means of which liquids can be taken up and distributed without leaving a residue. Liquid residues which can either be entrained further as contamination or which reduce the volume to be pipetted should not remain on the pipette or pipette tip.

- 4 - O.Z. 5496 - It is known from another technical area that structured surtaces repel liquids and are self-cleaning. Such surfaces and processes for their production are disclosed, for example, in DE 19 80 3787 or DE 19 91 4007, where it is described how drops run off structured surfaces as beads and can absorb dirt in the process.
There are significant differences between the detachment behavior and rolling-off behavior of a drop on a surface. In physical terms, rolling off 1 o always involves a component of gravity acting into the surface. Only when gravity points parallel to or even away from the surface can the drop detach. Furthermore, the rolling-off behavior is essentially described by the internal friction of the liquid. This is a function of the surface tension of the liquid and the interfacial tension between the liquid and the surface. Only the interfacial tension plays a significant role during detachment of the liquid drop. However, a precise physical description has still not been given (W. Zhang, Physical Review Letters, Vol. 83, pages 1151 ff., 1999).
Surprisingly, it has been found that structured surfaces whose structures 2o have a certain aspect ratio, i.e. a certain ratio between the height and mean width, improve the detachment of liquid drops.
The present invention therefore relates to pipettes having structured surfaces, where the surfaces of the pipettes which come into contact with a liquid have elevations having a mean height of from 50 nm to 10 Nm and a mean separation of from 50 nm to 10 Nm and surface energies of less than 19 mNlm.
The pipettes according to the invention can have completely structured 3 o surfaces or partly structured surfaces. It is important that the surfaces which come into contact with a liquid are completely or partly structured.
Commercially available pipettes or pipette tips are as smooth as possible inside and outside in order to facilitate rapid flow-off of liquids. The pipettes - 5 - O.Z. 5496 according to the invention, through a contrasting measure, namely the structuring of the surface, facilitate virtually complete emptying of the pipette and substantially splash-free detachment of even the smallest amounts of liquid.
The surtace energy of the structured regions, which is determined on unstructured material, is less than 19 mNlm, preferably from 10 to 18 mN/m, in the case of the pipettes according to the invention.
1 o The structured surfaces employed in the pipettes according to the invention are extremely hydrophobic and therefore have a strong water-repellent action. They have a very high contact angle with water and promote the detachment of liquid drops.
The contact angle or surface energy is advantageously determined on smooth, unstructured surfaces. The material properties "hydrophobicity", "liquid repulsion" and "liquid wetting" are also partly determined by the chemical composition of the uppermost molecule layers of the surface.
Higher or lower contact angles or lower or higher surface energy of a 2 o material can therefore also be achieved by coating processes.
The hydrophobic properties of a surface can thus be defined via the surface energy, with the contact angle of smooth, i.e. unstructured material with various liquids being a measure of the surface energy, which is quoted 2 5 in mN/m.
The pipettes according to the invention can also have elevations with different dimensions. The mean height of the elevation is from 50 nm to 4 Nm with a mean separation of from 50 nm to 10 pm. Alternatively, the 3 o mean height of the elevations can be from 50 nm to 10 pm with a mean separation of from 50 nm to 4 pm. The elevations particularly preferably have a height of from 50 nm to 4 Nm with a mean separation of from 50 nm to 4 Nm.

~ ~' - 6 - O.Z. 5496 The ratio between the height and width of the elevations, the aspect ratio, - is, as already mentioned, of considerable importance. The elevations can have aspect ratios of from 0.5 to 20, preferably from 1 to 10, particularly preferably from 1 to 5.
The chemical composition of the uppermost monolayer of the material is also important.
For changing the chemical surface properties, mention should be made of 1 o processes in which free-radical sites are generated on the surface. The structured or unstructured material can be treated by plasma, UV or gamma radiation and special photoinitiators. After surface activation of.this type, i. e. generation of free radicals, additional monomers can be polymerized on. A process of this type generates a coating which is particularly chemical-resistant. Examples of suitable monomers are acrylates, methacrylates and other vinyl derivatives, such as, for example, perfluorohexylethylene methacrylate.
The shaping or structuring of the surface can be carried out by 2 o embossing/rolling or simultaneously with macroscopic shaping of the article, such as, for example, casting, injection molding or other shaping methods. To this end, the corresponding negative molds of the desired structure are necessary. For the production of the pipettes according to the invention, an injection-molding process is advantageously employed.
Negative molds for, for example, injection-molding processes can be produced industrially using, for example, the LIGA technique (R. Wechsung in Mikroelektronik, 9, 1995, pp. 34 ff.), in which firstly one or more masks are produced with the dimensions of the desired elevations by electron 3o beam lithography. These masks are used for exposure of a photoresist coating by deep X-ray lithography, producing a positive mold. The interspaces in the photoresist are then filled by electrodeposition of a metal. The metal structure obtained in this way represents a negative mold for the desired structure.

" - 7 - O.Z. 5496 - In another embodiment of the present invention, the elevations are arranged on a somewhat coarser superstructure. The elevations have the abovementioned dimensions and can be applied to a superstructure having a mean height of from 10 Nm to 1 mm and a mean separation of from Nm to 1 mm. The elevations on the superstructure can likewise be embossed, or applied by lithographic methods or shaping processes, such as casting or injection molding. The elevations and the superstructure can be mechanically embossed or applied by lithographic methods or by 1 o shaping processes, such as casting or injection molding, simultaneously or successively, i.e. firstly the superstructure, then the elevations.
The shaping or structuring of the surfaces is advantageously carried out in a single operation in the case of surfaces having a superstructure, as in the case of surfaces having only elevations. Subsequent chemical modification of a double-structured surface which has already been produced is of course likewise possible.
Mechanical processes for introducing structures onto unstructured surfaces or unstructured areas of structured surtaces are, for example, embossing or stamping methods using prefabricated molds or stamps (needles).
Lithographic methods are, for example, the LIGA technique, X-ray lithography or alternatively ablative methods, for example using laser radiation.
It is likewise possible to produce a suitable surface energy by applying resists or other coating materials. The surtace energy can be set to the desired value in this process or subsequently. The prerequisite for this process is an inert behavior of the coating with respect to the liquids to be 3 o pipetted.
The structuring of the pipettes according to the invention is of course only necessary where the pipettes come into contact with a liquid. For a simple ,.
- 8 - O.Z. 5496 production process of, for example, pipette tips, the entire pipette surface can also be structured.
The structuring, i.e. the elevations, can be applied to the inside (a in Fig.
1 ) or outside surface of the pipette (b in Fig. 2). It is also possible to apply the elevations only to the pipette tip, i.e. to the pipette outlet (c in Fig. 3).
The materials used for the pipettes according to the invention must have the requisite values for the surface energy in the unstructured state. Use 1 o can be made, for example, of poly(tetrafluoroethylene), poly(trifluoroethylene), poly(vinylidene fluoride), poly(chlorotrifluoroethylene), poly(hexafluoropropylene), poly(perfluoropropylene oxide), poly(2,2,3,3-tetrafluorooxetane), poly(2,2-bis(trifluoromethyl)-4,5-difluoro-1,3-dioxole), poly(fluoroalkyl acrylate), poly-(fluoroalkyl methacylate), polyvinyl perfluoroalkyl ether) or other polymers of perfluoroalkoxy compounds, poly(ethylene), poly(propylene), poly-(isobutene), poly(isoprene), poly(4-methyl-1-pentene), polyvinyl alkanoates) and polyvinyl methyl ether) as homopolymers or copolymers, and other thermoplastically processable plastics.
Example:
The pipettes can be produced, for example, by an injection-molding process in combination with a conventional injection mold produced by the 2 5 LIGA process. The LIGA process is a structuring process based on the basic processes of X-ray lithography, electroplating and casting. The process differs from micromechanics in that the structures are not produced by an etching process in the basic material, but instead can be cast inexpensively via a mold. In the present case, the LIGA process is 3 o used to produce the mold. After lithographic resist exposure (radiation-sensitive polymer) and development, the resultant resist structure is used as the mold for an electroplating process, in which a metal alloy is deposited in the exposed interspaces. The resist structure is then removed, and the remaining metal structure utilized as a casting mold (G. Gerlach, - 9 - O.Z. 5496 W. Dotzel, "Grundlagen der Mikrosystemtechnik" [Fundamentals of Microsystem Technology], Carl Hanser Verlag, Munich, 1997, pages 60 ff.).
Pipette tips with a pipetting volume of from 0.5 to 10 NI with a microstructured surface at the outlet end were produced by injection molding from polypropylene) with the aid of a mold as described above (see Fig. 3). The separation between the elevations here was on average 4 Nm, with a height of least 4 Nm and a half-height width of 2 Nm. In geometrical terms, the structural elements were conical in shape.
The pipette tips were subsequently exposed to UV radiation at 254 nm for two minutes. Fluoroalkyl acrylate was thermally grafted onto the surfaces activated in this way. This procedure reduced the surface energy from about 28 rnNlm to less than 15 mN/m. In the case of unstructured and non-hydrophobicized pipette tips, the drops only detached at a pipette volume of 1.5 ml. In the case of the microstructured and hydrophobicized pipette tips according to the invention, this occurred at less than 200 n1. This could be demonstrated with liquids having different surface tensions and viscosities, such as, for example, deionized water, hexadecane, 10%
2 o aqueous albumin solution and DMSO.
Pipettes produced in this way can be used in automatic pipetting systems or dispensers.

Claims (12)

Claims:

1. ~A pipette having a structured surface, wherein the surfaces of the pipette which come into contact with a liquid have elevations having a mean height of from 50 nm to 10 µm and a mean separation of from 50 nm to 10 µm and surface energies of less than 19 mN/m.

2. ~A pipette as claimed in claim 1, wherein the elevations have a mean height of from 50 nm to 4 µm.

3. ~A pipette as claimed in claim 1, wherein the mean separation of the elevations is from 50 nm to 4 µm.

4. ~A pipette as claimed in claim 1, wherein the elevations have a mean height of from 50 nm to 4 µm and a mean separation of from 50 nm to 4 µm.

5. ~A pipette as claimed in one of claims 1 to 4, wherein the elevations have an aspect ratio of from 0.5 to 20.

6. ~A pipette as claimed in claim 5, wherein the elevations have an aspect ratio of from 1 to 10.

7. ~A pipette as claimed in claim 5, wherein the elevations have an aspect ratio of from 1 to 5.

8. ~A pipette as claimed in one of claims 1 to 7, wherein the elevations are applied to a superstructure having a mean height of from 10 µm to 1 mm and a mean separation of from 10 µm to 1 mm.

9. ~A pipette as claimed in one of claims 1 to 8, wherein the elevations are applied to the inside surface of the pipette.

10. A pipette as claimed in one of claims 1 to 9, wherein the elevations are applied to the outside surface of the pipette.

11. A pipette as claimed in one of claims 1 to 8, wherein the elevations are applied to the pipette outlet.

12. A pipette as claimed in one of claims 1 to 11, which consists completely or partly of poly(tetrafluoroethylene), poly(trifluoroethyl-ene), poly(vinylidene fluoride), poly(chlorotrifluoroethylene), poly(hexafluoropropylene), poly(perfluoropropylene oxide), poly(2,2,3,3-tetrafluorooxetane), poly(2,2-bis(trifluoromethyl)-4,5-difluoro-1,3-dioxole), poly(fluoroalkyl acrylate), poly(fluoroalkyl methacylate), polyvinyl perfluoroalkyl ether) or other polymers of perfluoroalkoxy compounds, poly(ethylene), poly(propylene), poly-(isobutene), poly(isoprene), poly(4-methyl-1-pentene), poly(vinyl alkanoates) and polyvinyl methyl ether) as homopolymers or copolymers.