NL1009704C2 - Preparation container and a method for manufacturing such a preparation container. - Google Patents

Description

Title: Preparation container and a method of manufacturing such a preparation container.

The invention relates to a method for manufacturing a preparation container, particularly suitable for use in shape-oriented research, in particular with an Atomic Force Microscope (AFM / SFM) or the like device.

It is usual for research of preparations to use a glass or mica carrier on which the preparation, in particular a biochemical preparation such as viruses, antigens or the like, is applied.

For this purpose, the glass or mica carrier is pretreated with a primer, such that active groups can be attached to it for binding the preparation. A glass or, in particular, a mica support has the advantage that it has a particularly smooth support surface, which is necessary to obtain a good resolution. After all, with a microscope it should be possible to observe particularly small elements on the support surface.

It is particularly difficult to suitably place such active groups on a glass or mica support. First, the adhesion to such a carrier is particularly difficult to achieve, which makes the carriers expensive. Secondly, it is not possible to obtain a good uniform, densely packed distribution of the active groups. This means that the resolving power of the microscope is considerably reduced by this preparation carrier, at least that it is not possible to make optimum use of the resolving power of the microscope, while moreover a relatively large chance of measuring errors occurs. A further drawback of mica or glass preparation carriers is that they are particularly fragile, which makes treatment difficult and increases the risk of damage and failure.

The invention contemplates a method for manufacturing a preparation carrier of the type described in the preamble 5, wherein the drawbacks of known methods and preparation carriers are avoided, while the advantages thereof are retained. To this end, a method according to the invention is characterized by the measures according to claim 1.

In principle, plastic is a favorable material for the preparation of preparation carriers in that it is relatively easy to process and relatively strong, while good binding of various preparations, in particular biochemical preparations such as viruses, antigens, peptides and the like, can be obtained therefrom. obtained. However, plastic has the drawback that its surface roughness has heretofore been found to be too high to be suitable for use as a preparation carrier for use in Atomic Force Microscope or the like form-oriented research.

Surprisingly, it has now been found that with a method according to the present invention such a smooth plastic surface can be obtained that it is, at least much more suitable as a bearing surface for preparations in such research. Due to the plastic layer, treated thermally or chemically, against Namely, to form a surface of a support base with a suitable surface roughness, it appears that as a result thereof the surface roughness of the surface lying against the support base can be considerably reduced, for example, a reduction of the surface roughness can be obtained by a factor of 5 to 20 or more. means that elements of a preparation that are bound to the carrier surface can have particularly small dimensions, while still being able to optimally determine the shape, for instance with an Atomic Force Microscope, or at least its presence can be determined on the basis of 1009 704 3 of at least the shape. For example, these elements may have dimensions on the order of 20 nm (viruses), or 3 nm (antibodies) or even smaller, on the order of 5, for example, 1 nm.

In a particularly advantageous embodiment, a method according to the invention is characterized by the measures according to claim 2.

Optimum distribution of the plastic can be obtained in a particularly simple manner by at least partially melting the plastic 10 against a surface of the carrier base. Moreover, it is easy to start from, for example, plastic film or plate. However, it is also possible, for example, to cause polymerization of the plastic layer on the support surface or to chemically treat the plastic in such a way that it liquefies against the surface of the support base.

Without wishing to be bound by any theory, the particular smoothness of the resulting support surface appears to be at least partly due to the use of a particularly smooth support base and the absence of adhesion to the support base. A method according to the present invention therefore seems to be optimized by using a carrier base with optimum smoothness and absence of adhesion between the plastic and the carrier base. However, even under sub-optimal conditions, sufficiently smooth carrier surfaces can already be obtained.

In a first preferred embodiment, a method according to the invention is further characterized by the measures according to claim 3.

The use of a plastic with at least one active group for the respective preparation offers the advantage that no post-processing is necessary. A group suitable for covalent binding of antigens thereby offers the advantage of 1009 704 4 that such a preparation carrier is particularly suitable for use in biotechnology.

In an alternative embodiment, a method according to the invention is characterized by the measures according to claim 4.

When the plastic used is not directly, at least not sufficiently suitable for binding the relevant preparation, it is preferred that the carrier surface is treated such that one or more active groups for the relevant preparation are applied to, at least in the carrier 10 'surface. , again especially groups for covalent binding of antigens such as a -COOH group. This achieves the advantage that a material can be used as plastic for the carrier surface, with particularly suitable properties for it, such as, for example, polyethylene, in particular high-molecular polyethylene, while the treatment of the carrier surface ensures that binding of the antigens is nevertheless becomes very possible. Plastic 20 has the advantage, above for instance mica and glass, that such a treatment is particularly simple and well possible, whereby a suitable treatment can always be selected, depending on the preparation to be bound.

In further elaboration, such a method is preferably characterized by the measures according to claim 5.

By inoculating the support surface with a plastic, a support surface which is not or insufficiently binding per se can easily be treated in order to obtain the desired bonding capacity, in particular covalent bonding. The use of acrylic acid or methyl acrylate is particularly suitable for this.

In a further advantageous embodiment, a method according to the invention is further characterized by the measures according to claim 8.

1009 704 5

Surprisingly, it has been found that the surface roughness of a support surface can, where appropriate, be further reduced by introducing -NH2 groups into, at least on the support surface. For example, the surface roughness of an polyethylene treated with acrylic acid or methyl acrylate can thereby be reduced such that it can still be made suitable, or at least better suited for use in Atomic Force Microscope research.

In further elaboration, a method according to the invention is further characterized by the measures according to claim 9, preferably by the measures according to claims 9 and 10.

By bringing a solution of a suitable monomer into contact with the carrier surface and then treating d% plastic and solution, such that polymerization of at least a part of the monomer occurs, a thin so-called adhesive layer can be obtained in a particularly simple manner, which is capable of effecting the desired compounds, especially covalent bonds, are applied to the support surface. This polymerization can be effected and monitored very well by means of suitable radiation. It is preferred that the polymerized adhesive layer has a relatively small thickness, in order to maintain good adhesion and a sufficiently flat surface.

As a carrier base, particularly suitable are surfaces formed from, for example, mica or glass or materials with comparable surface roughness, hardness and / or porosity. Mica in particular appears to be particularly suitable for this, especially because of its substantially atomic flatness.

The invention further relates to a preparation carrier for use in mold research, for example with a Atomic Force Microscope or the like, characterized by the measures according to claim 13.

Precisely a preparation carrier with a carrier surface made of plastic, with a surface roughness such that viruses or antibodies or the like biochemical elements incorporated thereon can be observed with an Atomic Force Microscope or such a device offers the advantage that such a preparation carrier is particularly is easy to manufacture and adapt to the preparations to be examined, while such a preparation carrier can be used in a particularly simple manner, in particular also because it is relatively strong.

Because the carrier surface is suitable for covalent bonding of the preparation, the advantage is thereby achieved that during use non-covalently bonded and / or non-bonded elements of the preparation can be easily washed away or otherwise treated, so that all kinds of tests known per se on the preparation can be carried out. It is precisely the specific binding of elements of the preparation with specific active groups of the carrier surface that makes these tests possible.

In further elaboration, a preparation carrier according to the invention is further characterized by the measures according to claim 17.

-C00H groups in, at least on the surface, allow binding of antigens thereto in a simple manner using, for example, soluble carbodiamide (EDC) / sulfo-N-hydroxy-succinimide (sulfo NHS). -NH2 groups 30 additionally enable coupling of antigens via, for example, glutardialdehyde (GDA). The chemicals used depend on the desired bonds and are immediately clear to the skilled person. Of course, in other cases, other active groups can also be applied in or on the carrier surface.

i 1009 704 7

The invention further relates to the use of an Atomic Force Microscope or like devices for biochemical research, characterized by the features of claim 18.

By using such a preparation carrier, the advantage is achieved that research with an Atomic Force Microscope can be carried out faster and easier, with a relatively higher resolution, while a large number of antigens can be bound on a particularly small surface. Moreover, optimum use can be made of the resolving power of the microscope in question. Incidentally, it is noted that in addition to an Atomic Force Microscope, any suitable scanning method can be used, whether or not using direct contact, depending on the desired results.

Further further exemplary embodiments of methods and preparation carriers according to the invention are given in the further subclaims.

For clarification, exemplary embodiments of a method and a preparation carrier will be explained in more detail below with reference to the drawing. In the drawing: Fig. 1 shows a carrier base; Fig. 2 shows a carrier base with a plastic layer applied thereon; Fig. 3 shows the plastic layer detached from the carrier base; Fig. 3a shows a plastic layer according to Fig. 3, in an alternative plastic.

Fig. 4 shows the plastic layer with an adhesive layer grafted onto the support surface; Fig. 5 schematically shows a preparation carrier with antigens adhered to the carrier surface; Fig. 5a schematically shows a chemical bond of a PPV virus to the carrier surface; 1009 704 FIG. 6 are enlarged views of the surface of high molecular weight polyethylene, the surface of a blown or drawn polyethylene film, a support surface of polyethylene formed against a support base of mica and a surface of glass, respectively; Fig. 7 shows the surfaces of eight anti-mica shaped carrier surfaces of polyethylene, treated in various ways with a solution of the relevant monomer and specific intensity of radioactive radiation; and FIGS. 8a and 8b, respectively, a mica-formed polyethylene support surface, treated with acrylic acid, to which Porcine Parvo Virus and Plum Pox Virus are bound, respectively.

In this description, like or corresponding parts have like or corresponding reference numerals. Furthermore, as an example in this specification, unless stated otherwise, a preparation carrier suitable for covalently binding antigens on a carrier surface, made of treated against a mica-melted polyethylene, is started from. It will be clear, however, that other plastics and a different carrier base can also be used, for example a carrier base of glass and a polypropylene, polycarbonate, acrylic acid or methyl acrylate as plastic for the actual preparation carrier. The latter plastics in particular can offer the advantage that direct covalent bonding to them is possible. Polyethylene is relatively inert. Polyethylene however offers the advantage that it is relatively hard and strong, without being brittle. In addition, other plastics can be easily grafted onto this.

In this description, a relative flatness measure will always be used, whereby the maximum height (Z-axis) of protrusions above a nominal reference plane is given as a percentage of one of the 1009704 9 horizontal dimensions (X-axis) of the scanned surface. This horizontal measure is in the order of magnitude of 2-4.5 micrometers in this description. The measure of flatness V is therefore expressed in the following formula: Z axis - X 100% X axis

Examples of the flatness V of materials: 10 - mica: V = 0.1%; glass: V = 0.3% (Fig. 6d); high molecular polyethylene: V = 10% (Fig. 6a); polyethylene foil; V = 3% (Fig. 6b); and a polyethylene surface formed according to the invention, 15 V = 0.6% (Fig. 6c).

These dimensions and values are given by way of example only and should not be construed as limiting in any way.

20 Legend: In the drawing holds: □ = -C00HO = -NH2 £ ƒ = virus or antibody. 1 2 3 4 5 6 1009 704

Fig. 1 shows in cross-sectional side view a carrier base 2, formed of mica, with an upper surface 43 with a flatness V of about 0.1%. This therefore means 4 that there are 4 irregularities on the plane with a maximum height in the Z direction measured above the nominal plane N of not more than a few nanometers, for example 4 to 5 nanometers. The surface 4 of mica is therefore particularly flat. The surface 4 is, for example, rectangular with outer dimensions of 25 x 25 millimeters. The base carrier 2 has a thickness of, for example, 0.5 millimeters.

10

In the state shown in Fig. 2, a plastic layer 6 is applied to the smooth top surface 4 of the base carrier 2. In the embodiment shown, this is a polyethylene film as shown in Fig. 6b, with an inherent flatness of about 3%. The foil layer has a thickness of, for example, 0.035 millimeters.

The foil layer 6 and / or the base carrier 2 are heated such that at least the side of the plastic layer 6 facing the surface 4 melts and flows on the surface 4, after which the whole is cooled. No significant adhesion will occur between the mica base carrier and the plastic layer 6, as a result of which the plastic layer 6 can easily be removed again from the base carrier 2. Surprisingly, it has been found that the surface 8 of the plastic layer 6 facing the base support 2 has acquired a flatness V which is considerably better than the flatness V of the polyethylene film used. For example, the flatness of the support surface 8 is about 0.6% if no further special measures have been taken.

Incidentally, it is noted that liquefaction of at least the part of the plastic layer 6 facing the base support 2 can also be obtained, at least partly, for instance by a chemical reaction.

FIG. 3 shows a preparation carrier 1 formed according to the present invention, the carrier surface 8 facing upwards. In this embodiment shown, polyethylene is used as plastic, which is relatively inert. Binding, in particular covalent bonding of biochemical elements thereto, is therefore in fact impossible. In fig. 3a an alternative embodiment is shown, wherein as plastic layer 106 a plastic is used with active groups 112 therein, symbolically represented by balls placed on sticks. Such a plastic can for instance be a polycarbonate, an acrylic acid or methyl acrylate. 1009704 11 in which, for example, as active groups 112 -COOH or -NH2 groups are present, symbolically shown in the drawing with a square and a ball on a stick, respectively.

Fig. 4 shows a preparation carrier 1 with a plastic layer 10 grafted thereon, for example a polymerized layer of acrylic acid or methyl acrylate. Such a layer 10 can be applied to the plastic carrier layer 6 of polyethylene or other plastic as follows.

The plastic part 6 with the smooth support surface 8 is immersed in a solution of a monomer of a specific concentration, after which the solution with the plastic incorporated therein is irradiated with radioactive radiation of a specific intensity, such that at least on the support surface 8 polymerization of the relevant monomer occurs.

Suitable monomer solutions are, for example, a 0.6% or 6% acrylic acid (AC) monomer solution or a 0.6% or 6% methyl acrylate (MA) monomer solution. These can, for example, be irradiated with γ radiation of, for example, 2 or 12 kilos of Gray (kGy). By a suitable choice of the irradiation period, a desired thickness of the respective polymerized layer on and partly in the support surface 8 is thus obtained. Such a layer is preferably as thin as possible, for instance a few molecules or chains thick, so that the flatness of the support surface 8 is retained as much as possible or even increased.

Fig. 7 shows eight preparation carriers according to Fig. 4, grafted in solutions of monomers acrylic acid or methyl acrylate with different concentrations and different irradiation intensities. As can be seen from Fig. 7, in particular the surfaces shown in Figs. 7c, 7d and 7h are particularly flat and are therefore extremely suitable for specimen testing. The coding successively shows the carrier plastic (PE), the 1009 704 12 concentration of the solution (in%), the amount of irradiation (in kilograms of Gray) and the graft plastic used (AC or MA). Other combinations are of course also possible, for instance more or less or different monomers, other irradiation amounts, other polymerization methods and other carrier plastics. Suitable choices are immediately clear to the skilled person and can be determined without further invention.

The viruses or antibodies to be bound have or are provided with active groups, for example -COOH groups and / or -NH2 groups, which can be linked directly or via linkers to the active groups 12 on, at least in the support surface 8, 10. So For example, -NH2 groups of a virus can be linked to a -COOH group or a -NH2 group of the support surface 8, 10, while -COOH groups of a virus can be linked to -NH2 groups of the support surface 8, 10, for example. Various chemicals can be used as linkers, for example HMDA 20 (Hexamethylenediamine) or EDA (Ethylenediamine). Thus, for example, -NH2 groups as active groups can be introduced into or on a support surface 8, 10 comprising only or substantially, for example, -COOH groups as active groups 12. HMDA can be used by coupling Boe HMDA (Butyloxycarbonylhexamethylenediamine) via DCC (Dicyclohexylcarbodiimine) to the -COOH groups, making -NH2 groups available for antigen coupling after Boc deprotection. When using EDA, a surface treated with methyl acrylate 8,

Follow a treatment of, for example, 72 hours at 40 ° C

with said EDA, whereby active -NH2 groups become available. For example, the first support surfaces are PE (0.6 / 2 Ac) Hmda and PE (0.6 / 12Ac) -Hmda, while the second type of surface conforms to PE (0.6 / 2 MA) -EDA, for example.

The other surfaces shown in Fig. 7 are less flat. Introduction of -NH2 groups in these surfaces- 1009 704 13 surfaces, for example in the manner described above, surprisingly leads to an improvement in the flatness V of these surfaces. This means that, by introducing said -NH2 groups therein, these surfaces also become, at least even better, suitable for use as a preparation carrier for at least form-oriented research.

Fig. 5 schematically shows how a number of viruses 14 are linked to the active groups of a preparation carrier 1. Likewise, coupling to a 10 'preparation carrier 101 as shown in Fig. 3A is possible. Fig. 5A schematically shows how chemical binding of a PPV virus to a carrier surface is possible. Antigens and the like can be coupled to the resulting surfaces with -COOH groups via, for example, EDC (soluble carbodiamide) / sulfo NHS (sulfo-N-hydroxy-succinimide), known from the Pierce catalog. Antigens and the like can be coupled to the resulting surfaces with -NH2 groups, for example via glutar-dialdehyde (GDA). The latter coupling is shown in Fig. 20 5A and in Fig. 8 and may be referred to as PE (0.6 / 2Ac) -Hmda-Gda-PPV- (Porcine Parvo Virus) or - (Plum Pox Virus).

A preparation carrier according to Fig. 5, with viruses 14 or the like bound thereto, can for instance be examined under an Atomic Force Microscope or the like device, wherein the presence and / or the shape of the bound viruses 14 can be accurately determined due to the high flatness of the carrier surface 8. From the examples shown in fig. 8 it can be seen that, for example, a distinction can be made between the relatively spherical PPV (fig. 8a) and the substantially string-shaped Plum Pox Virus. (fig.8b)

A preparation carrier according to the present invention offers an important advantage over the prior art that in a particularly simple manner 35 different types of active groups can be applied, at least in the carrier surface, such as the 1009704 14 -COOH groups and -NH2 groups mentioned. . Depending on the desired application and the desired bonds, the support surface can, if necessary, be treated appropriately. In addition, the active groups can be applied particularly close to one another, so that a high density of the elements to be detected from the preparation can be obtained, for example with an occupancy rate of 10% or more. Occupancy rates of, for example, 70-100% are possible. As a result, the resolution of the detection technique used can be considerably increased, or at least more optimally utilized.

The flatness of the support surface 8 can optionally be increased even further by making use of appropriate techniques, for example vacuum techniques 15 when placing and melting on the support base 2, or at least causing the plastic layer 6 to flow. This prevents gas inclusions from becoming uneven lead.

The invention is by no means limited to the exemplary embodiments shown in the drawing and the description. Many variations thereof are possible within the framework of the invention outlined by the appended claims.

For example, other plastics can be used to form the support surface and / or to graft the layer 10. Suitable plastics can be selected, for example, on the basis of the desired active groups, the desired hardness or flexibility, the desired combination of carrier plastic and graft plastic, any resistance to, for example, chemicals, irradiation, exposure and the like. Such choices will be readily apparent to those skilled in the art within the scope of the invention.

Furthermore, preparation carriers according to the present invention can also be used for other studies, for example using markers for determining the presence of certain elements, eg fluorescent, coloring or radiant markers. In the exemplary embodiments shown, the plastic layer is always applied to the base carrier, but it is of course also possible to process a plastic layer with a sufficiently smooth surface of a base carrier which is moved against or along the surface of the plastic layer, for example a base carrier of mica or glass. It is also possible to cause polymerization of a plastic to take place on a base support with the desired smoothness or to otherwise obtain plastic on it with suitable properties. Of course, all kinds of different preparations can be bound on a preparation carrier according to the present invention. The viruses described serve only as an example.

These and many comparable variations are considered to fall within the framework of the invention outlined by the claims.

1 0 0 9 7 04

Claims (18)

A method of manufacturing a preparation support for use in preparation research, in particular shape-oriented research, such as Atomic Force Microscope (AFM, SFM) research, wherein: 5 a layer of plastic is applied to at least one surface of a support base wherein the plastic layer is thermally and / or chemically treated, such that the surface roughness of the side of the plastic facing the carrier base is reduced, while it hardly adheres to the carrier base, after which the plastic is removed from the carrier base, the released, relatively smooth surface of the plastic forming a support surface. A method according to claim 1, wherein the plastic is applied by at least partial melting over the at least one relevant surface of the carrier base. 3. A method according to claim 1 or 2, wherein as a plastic a mono- or polymer is used with at least one group active for the preparation in question, in particular a group for covalent binding of antigens such as a -COOH group. 4. A method according to claim 1 or 2, wherein the support surface is treated such that the support surface comprises at least one group active for the respective preparation, in particular a group for covalent binding of antigens such as a -COOH group. Method according to claim 4, wherein the carrier surface is inoculated with a plastic, in particular with the aid of a mono- or polymer, preferably acrylic acid or methyl acrylate. The method of claim 5, wherein the support surface is treated with acrylic acid or the like and 1009 704 is further treated to obtain -NH 2 groups thereon, preferably by coupling thereto hexamethylenediamine (Hmda). The method of claim 5, wherein the support surface is treated with methyl acrylate or the like and further treated to obtain -NH 2 groups thereon, preferably using ethylenediamine (EDA). 8. A method according to any one of claims 5-7, wherein the surface roughness thereof is reduced by introducing -NH2 groups in, at least on the support surface. 9. A method according to any one of claims 4-8, wherein at least the plastic layer on at least the support surface is brought into contact with a solution of a monomer, after which the plastic and the solution are treated such that polymerization of at least a part of the monomer occurs on the support surface, for which purpose the plastic is preferably exposed to radiation together with the solution. A method according to claim 9, wherein the support surface is provided with a polymerized adhesive layer of a relatively small thickness, preferably a thickness of at most a few atoms or relatively flat chains. 11. A method according to any one of the preceding claims, wherein a carrier base is used with a particularly low surface roughness of at least the surface on which the plastic is applied, preferably with a surface roughness of the order of atomic roughness or slightly above it. A method according to claim 11, wherein a base support is used, at least said surface of which is made of mica or glass or a material comparable in surface roughness, hardness and porosity, preferably of mica. 13. Preparation support, in particular for use with Atomic Force Microscope (AFM, SFM) or the like form-oriented 1009704 examination of a preparation, in particular a biochemical preparation, which preparation support has a support surface made of plastic, the support surface having a surface roughness has such that viruses or antibodies or like biochemical elements incorporated thereon are detectable with an Atomic Force Microscope (AFM / SFM) or the like device, the support surface being suitable for at least covalently binding the composition. A preparation carrier according to claim 13, wherein the carrier surface is formed by at least partially melting the plastic on a carrier base with a surface roughness less than or approximately equal to the surface roughness of the carrier surface. Preparation carrier according to claim 13 or 14, wherein the plastic is a polymer, in particular polyethylene or polypropylene. 16. Preparation carrier according to any one of claims 13-15, wherein the carrier surface is grafted using a mono- or polymer, preferably acrylic acid or methyl acrylate. Preparation carrier according to any one of claims 13-16, wherein the carrier surface comprises at least -C00H groups and preferably -C00H groups and / or -NH2 groups. 18. Use of an Atomic Force Microscope (AFM, SFM) for biochemical research, wherein a preparation carrier is provided with a carrier base and a plastic carrier surface, preferably according to any one of claims 13-17, wherein antigens are covalently bonded to the carrier surface. 1009704