EP2292329B1 - Polymer substrate with fluorescent structure, method for production of same and application of same - Google Patents

Description

The invention relates to polymer substrates provided with fluorescence features in which fluorescence characteristics, ie fluorescent structures, are produced photochemically by UV irradiation. For example, in polymer substrates with a low intrinsic fluorescence, suitable fluorophors can be produced by suitable UV irradiation, which exhibit marked and detectable emission upon excitation with light of suitable wavelength. If such irradiation is carried out in a structured manner, emission patterns can be generated in this way in polymer substrates, which can be used, for example, as a recovery grid in fluorescence microscopy. Another field of application relates to product authentication, which is made possible by the polymer substrates provided with fluorescence features according to the invention.

In the field of culturing cells, a wide variety of sample chambers are used. These are mostly polymer-based and range from the cell culture bottle to slides and μ-slides from ibidi to multiwell plates.

These sample chambers can have contiguous areas on which cells grow from 1 mm ² to 100 cm ² . Since cells typically have a diameter of 1 .mu.m to 30 .mu.m, finding individual cells in large-area chambers without recovery structures is almost impossible.

So is out of the DE 100 04 135 a recovery grid on a plastic film, wherein the plastic film is integrated into the sample chamber. The EP 2 008 715 A1 also describes a recovery grid, which is formed as part of the plastic body or is introduced into the plastic body. The systems described here are based on non-fluorescent recovery gratings.

Another area of the prior art underlying the present invention relates to the authentication of products, especially consumer products.

The authentication of products currently takes place in the prior art by the application of fluorescent material on polymer films by means of various printing processes. Other methods of authenticating products provide for the characterization of the intrinsic fluorescence of polymers, with the preparation of the polymers having added fluorescent dyes as dopants. ( WO 2005/054830 ).

Furthermore, in fluorescence films can be structured by bleaching existing autofluorescence.

Likewise, methods for structuring fluorescence are known in which polymers are used which contain covalently bonded precursors or to which such must be added as dopants.

Likewise, more complex systems are known which use fluorescence as a means of authentication, but with changes in the shape of the film combinations are required ( WO 2003/101755 ). Another variant provides that the anisotropy of fluorescence dopants in stretched films is exploited in order to produce authentication materials ( WO 2004/087795 ). All these methods for the authentication of products have in common that the fluorophores are distributed evenly throughout the material so that there are no structured areas of fluorescence.

The FR 2 755902 A1 relates to a method for producing a labeled product as an integral component, wherein the label is caused by a fluorescent structure.

In QI C.-L. ET AL .: "Blue light emission from pulsed laser ablated polyethylene" OPTOELECTRONICS LETTERS, Vol. 4, No. 1, January 2008 (2008-01), it is described that a fluorescent structure can be produced by subjecting the surface of polytetrafluoroethylene to irradiation with a focused pulsed laser, whereby a fluence oreszenzemission the evaporated PTFE areas could be determined.

In CARLSSON DJ ET AL .: "Photooxidation of polypropylene films.""Origin of preferential surface oxidation." MACROMOLECULES, Vol. 4, No. 2, 1971, pages 179-184 the photooxidation of polypropylene films is described.

Out BAKER AK ET AL .: Refractive index modification of polymethylmethacrylate (PMMA) thin films by KrFlaser irradiation APPLIED PHYSICS A (SOLIDS AND SUR-FACES), Vol. A57, No. 6, December 1993 (1993-12), pages 543 -544, Germany ISSN: 0721-7250 the use of polymers for optoelectronic and optical applications is known. It is further described that the laser irradiation of PMMA leads to a strong increase in light absorption in the UV range.

In US 2004/055492 A1 describes a method for marking polymers, wherein the polymer has a component which is irreversibly transferred by irradiation with laser light in a second component, so that a visible contrast of the polymer takes place.

Out FR 2 909 922 A1 For example, a method is known for marking various materials, such as metal, plastic or ceramic, for example, wherein the marking is generated by laser bombardment in the picosecond range.

The DE 42 41 663 A1 relates to a method of marking an article, wherein the article is provided with a bar code which is readable by means of visible or UV light.

Based on this, it was an object of the present invention to provide fluorescent polymer substrates, which are easy to prepare and allow easy detection or detection.

This object is achieved by the polymer substrate having the features of claim 1 and the process for its preparation having the features of claim 17. In claims 24 to 28 uses according to the invention of the polymer substrate are listed. Likewise, a sample chamber according to claim 7 be provided having this polymer substrate. The other dependent claims show advantageous developments.

According to the present invention, there is provided a polymer substrate having a fluorescent structure as an integral part, wherein the fluorescent structure is a photochemically generated recovery grid. The preferred polymer substrates are low intrinsic fluorescence polymers, i. with an intrinsic fluorescence on the order of a cover glass in the excitation and emission range of 200 nm to 1000 nm. These include in particular COC, COP, PMMA, aliphatic polyesters and polyurethanes and polyethers. Particularly good polymers are suitable without UV stabilizers. For optical microscopy, in particular polymers with a refractive index between 1.4 and 1.6, in particular with 1.51 and / or a Abbe number over 50 and / or with a low birefringence are suitable.

A fluorescent structure is understood to mean structures up to the optical resolution limit. Typically, however, structures with a lateral resolution of several μm are used. Preferably, the fluorescent structure should have at least twice as high intensity as the non-fluorescent structure. For classical applications, the structure should have such a high intensity that it can be easily recognized even at exposure times of less than one second with commercial fluorescence microscopes. This is already the case with a factor of 10.

According to the invention, the generated fluorophores, the form the fluorescent structure, firmly bound in the polymer substrate and form a unit with it. The fluorescent structures are thus an integral part of the carrier. The modified polymer is characterized by the fact that both the fluorescent regions and the non-fluorescent regions consist of the identical original material. Therefore, the fluorophores also can not diffuse or sweat out. These properties clearly delimit the structures produced, for example, from a printed, otherwise applied structure (eg an embossed or lasered structure) or dye-doped polymer systems. In addition, no, possibly cell toxic, fluorescent dyes have to be used, which are applied to the polymer.

According to the invention, a structuring of the polymer substrate with fluorescent regions is produced. For this purpose, a mask or a locally positionable radiation source is preferably used. If the UV irradiation is carried out in a structured manner, only the areas that have been irradiated will fluoresce significantly. In the non-irradiated areas, however, no substantial increase in the base emission is achieved. In this way, patterns can be generated which become visible upon excitation with light of suitable wavelength. Such a photochemical structuring produces patterned fluorescent areas that are long-term stable and stable to environmental influences. Furthermore, they are characterized by the fact that they do not lose their edge sharpness by diffusion or similar processes. In addition, the features thus produced can not be removed without trace.

Preferably, the fluorescent structure in the polymer substrate consists of several elements in the form of bars, lines, characters, figures, interference patterns, or combinations thereof.

As described above, fluorescent structures can be generated with only limited by the substrate size limited overall extent of the pattern. The lateral resolution of the elements of the fluorescent structure is limited only by the method of introduction and the wavelength of the light. However, the individual structural elements of the fluorescence pattern typically have a lateral extent of less than 100 μm, preferably less than 10 μm.

According to the invention, a method is also provided for producing the polymer substrate described above, in which a polymer substrate is at least partially subjected to irradiation in the wavelength range below 300 nm to produce fluorescent structures.

According to a variant of the invention, the preparation of the fluorescent structures by UV irradiation using masks by imaging or contact exposure takes place.

As a mask for structuring, it is preferable to use plates which have an exemplary variation of regions that are either transparent or non-transparent to the irradiation light used. Preference is given to using metallic plates which have patterned openings. Particularly preferred are masks made of chrome on silica glass.

As an alternative to masks, both radiation sources with coherent (laser direct writing) and focused beam (sample positioning) can be used for structuring.

Radiation sources used are preferably radiation sources with emission wavelengths or emission wavelength ranges in the range below 300 nm. These include, in particular, deuterium lamps, excimer lamps (Xe), excimer lasers (F ₂ , ArF, KrF) or solid-state lasers (Nd: YVO ₄ / YLF).

The polymer substrate used is preferably a polymer film.

The UV-generated generation of the fluorophores can be monitored in the absorption spectrum of the polymer substrate by an increase in the extinction in the UV range. However, these changes do not significantly affect the optical transmissivity in the visible region of the light to the human eye. The polymer substrates can be provided without damage on the front or back with fluorescent features. Their production requires no exclusion of atmospheric oxygen.

Due to a high range of absorption and emission of the generated fluorophores, different fluorescence colors can be generated according to the excitation conditions.

The fluorescent features are further characterized by the fact that they are generated directly in materials that are used anyway for products from different areas. There are no additional coatings, imprints or other application fluorophores, fluorescent labels or their precursors. The fact that the features are produced directly in the material used, makes it much easier and cheaper to manufacture this compared to other methods. In addition, it is a fully optical process that does not require any wet-chemical development processes.

Such emission patterns can be used to provide substrates for fluorescence microscopy with a recovery grid. Typically, the recovery grids are designed as a grid, which is also provided with numbers or letters. These recovery grids facilitate the retrieval of certain sample areas by being precisely charac- terised by the grid grid. The recovery grids do not significantly affect biological processes and are sufficiently stable to perform longer observations on biological and other samples with greater reliability and reproducibility.

Due to their absorption and emission properties, the described, photochemically generated characteristics are very well suited for typical excitation conditions in fluorescence microscopy; they are visible in all fluorescence channels (blue, green, yellow and red). The lateral resolution in such produced features is sufficiently high, e.g. to allow a mapped observation of cells of different types.

Chambers in which such gratings can be introduced are in the DE 100 04 135 , of the DE 101 05 711 , of the EP 02 777 215 , of the EP 05 041 563 , of the EP 06 015 167 , of the EP 07 012 400 and the EP 09 006 487 described.

These are essentially chambers which consist of at least one upper part and one lower part. In this case, the upper part has at least one recess. By connecting to the lower part of a reservoir is formed. Depending on the geometry of the recess of the upper part of the reservoir is then designed as a closed channel / tube or as an open-topped container.

The bottom formed by the base may be a foil or a coated glass carrier. The bottom has a preferred thickness of 50 .mu.m to 250 .mu.m and / or an Abbe number greater than 50 and / or a refractive index between 1.2 and 1.8, in particular between 1.45 and 1.55. These properties are particularly well suited for (high-resolution) microscopy. The bottom or film can be irradiated both from the side on which the cells are to be cultivated later and from the corresponding opposite side in order to produce fluorescent structures. When using high-resolution microscopy techniques with a correspondingly small depth of field (approximately less than 50 μm, typically less than 10 μm), it is advantageous to fluorescently mark the side on which the cells to be recovered will later grow. As a result, the cells and the lattice are simultaneously in optical focus, the fluorescent element being an integral part of the polymer carrier.

If the opposite side is irradiated, already covered microfluidic analysis chambers can be provided with corresponding features.

It is also possible to use composite films of different materials in which at least one component has the desired properties.

In particular, coated glass supports can also be used. Thus, e.g. Glass cover glasses are coated with a COP or COC layer to then introduce into this layer via the method described fluorescent grating. The layer may e.g. be applied by spincoating etc.

Another field of application concerns product authentication. Thus, labels, labels and products made of polymers can be provided with a feature by the structured UV irradiation, which becomes clearly visible only by excitation with light of suitable wavelength and intensity. In this way, the counterfeiting security of the product increases and a non-erasable product individualization can be carried out, for example via barcodes. This can be realized by a laser marking system, wherein the feature can also be read electronically.

With reference to the following figures and examples, the object according to the application is intended to be explained in more detail, without wishing to restrict it to the specific embodiments shown here.

Fig. 1 shows an emission spectrum of a polymer substrate according to the invention before and after the generation of fluorescence regions. The irradiation for the generation of the fluorescence regions took place here with an ArF excimer laser. The irradiation time was 10 seconds. By the irradiation is a clear Increase in absorbance in the wavelength range below 300 nm and strong increase in the emission from 400 to 600 nm.

In Fig. 2 a polymer substrate according to the invention is shown which has number patterns which were produced with the aid of a mask. This is a fluorescence micrograph under excitation at a wavelength of 365 nm and 200x magnification.

example 1

On a COC film, a mask (pattern chrome on silica glass) is attached, so that the mask rests with the chrome side. It is irradiated with a 30W deuterium lamp for 3 hours. Then the mask is removed and the emission pattern can be visualized by excitation at 365 nm.

Example 2

On a COC film, a mask (pattern chrome on silica glass) is attached, so that the mask rests with the chrome side. It is irradiated with an ArF excimer laser (193 nm) for 10 seconds. The mask is then removed and the emission pattern can be visualized by excitation at 365 nm, 436 nm or 515 nm.

Claims (28)

1. Polymer substrate having a fluorescent structure as an integral component, wherein the fluorescent structure is a photo-chemically generated recovery grid.
2. Polymer substrate according to claim 1, characterised in that the polymer substrate before the photochemical treatment and the starting materials for the production of the polymer substrate contain fundamentally no fluorophores or precursors thereof.
3. Polymer substrate according to claim 2, characterised in that the polymer substrate has fluorescent and non-fluorescent regions, wherein the fluorescent and non-fluorescent regions of the polymer substrate consist of the identical starting materials.
4. Polymer substrate according to one of the preceding claims, characterised in that the intensity of the emission of the fluorescent regions doubles, in particular is 10 times as high as the intensity of the emission of the non-fluorescent regions.
5. Polymer substrate according to one of the preceding claims, characterised in that the polymer substrate is selected from the group consisting of cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polymethyl methacrylate (PMMA), aliphatic polyesters, polyurethanes, polyethers or composites hereof.
6. Polymer substrate according to one of the preceding claims, characterised in that the fluorescent structures consist of several elements in the form of dashes, lines, characters, interference patterns or combinations hereof.
7. Polymer substrate according to the preceding claim, characterised in that the elements have a resolution that is sufficient for information coding of 1µm to 1000mm, preferably 1µm to 1mm, particularly preferably 1µm to 100µm.
8. Sample chamber containing a polymer substrate according to one of the preceding claims.
9. Sample chamber according to claim 8, characterised in that the fluorescent structure and the objects/cells to be recovered are situated in a focal plane.
10. Sample chamber according to one of claims 8 or 9, characterised in that the recovery grid and the objects/cells to be recovered are separated from one another spatially in the sample chamber.
11. Sample chamber according to one of claims 8 to 10, characterised in that the sample chamber has a base plate and a cover plate, wherein a recess is provided in the base plate, such that a receiving region is formed by the base plate and the cover plate, wherein the polymer substrate is provided in the cover plate.
12. Sample chamber according to claim 11, characterised in that the recess in the base plate has a base, such that a hollow space is formed by the cover plate.
13. Sample chamber according to claim 12, characterised in that the base plate and/or cover plate has a channel flowing from outside into the receiving region, in particular a through hole.
14. Sample chamber according to one of claims 11 to 13, characterised in that the cover plate has a thickness of 50pm - 250mm, in particular 100µm - 200pm.
15. Sample chamber according to one of claims 11 to 14, characterised in that the cover plate is formed as a film.
16. Sample chamber according to claim 15, wherein the fluorescent structure is introduced into the film.
17. Method for the production of a polymer substrate according to one of claims 1 to 7, in which a polymer substrate undergoes irradiation, at least in regions, in the wavelength range below 300nm for the generation of fluorescent structures.
18. Method according to claim 17, characterised in that fluorescent structures are generated from several elements in the form of dashes, lines, characters, interference patterns or combinations hereof.
19. Method according to one of claims 17 or 18, characterised in that the elements are generated with a resolution sufficient for information coding of 1µm to 1000mm, preferably from 1µm to 1mm, particularly preferably from 1µm to 100µm.
20. Method according to one of claims 17 to 19, characterised in that fluorescent structures are generated by UV-irradiation using masks by imaging or contact exposure.
21. Method according to claim 20, characterised in that plates having a pattern of transparent and non-transparent regions are used as masks.
22. Method according to one of claims 17 to 19, characterised in that fluorescent structures are generated using positioning systems by irradiation with coherent or focused light.
23. Method according to one of claims 17 to 22, characterised in that radiation sources having emission wavelengths or emission ranges of below 300nm are used for the structured generation of the fluorescent structures, in particular selected from the group consisting of deuterium lamps, excimer lamps (Xe), excimer lasers (F₂, ArF, KrF) or solid-body lasers (Nd: YVO₄/YLF).
24. Use of the polymer substrate according to one of claims 1 to 7 in a sample chamber.
25. Use of the polymer substrate according to one of claims 1 to 7 as a label or as tags for product authentication or product individualisation.
26. Use according to claim 25, characterised in that the fluorescent features contain information for product authentication or/and product individualisation in a coded or uncoded manner.
27. Use of the polymer substrate according to one of claims 1 to 7, in particular in the form of films, blister packaging and hollow bodies, as packaging elements.
28. Use according to one of claims 24 to 27, wherein the fluorescent features contain information in machine-readable form, in particular bar codes and dot patterns.

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