WO2000005583A1

WO2000005583A1 - Preparation carrier and method for its manufacture

Info

Publication number: WO2000005583A1
Application number: PCT/NL1999/000467
Authority: WO
Inventors: Wouter Cornelis Puijk
Original assignee: Stichting Dienst Landbouwkundig Onderzoek
Priority date: 1998-07-21
Filing date: 1999-07-20
Publication date: 2000-02-03
Also published as: AU5071499A; NL1009704C2

Abstract

Method for manufacturing a preparation carrier for use in preparation examination, in particular form-directed examination, such as Atomic Force Microscope (AFM, SFM) examination, wherein: on at least one surface of a carrier base, a layer of plastic is provided, wherein the plastic layer is treated thermally and/or chemically, such that the surface roughness of the side of the plastic that faces the carrier base is reduced, while it substantially does not adhere to the carrier base, whereupon the plastic is removed from the carrier base, with the released, relatively smooth surface of the plastic forming a carrier surface.

Description

PREPARATION CARRIER AND METHOD FOR ITS MANUFACTURE

The invention relates to a method for manufacturing a preparation holder, in particular suitable for use in form- directed examination, in particular with an Atomic Force Microscope (AFM/SFM) or a like apparatus. For the examination of preparations, it is conventional to use a glass or mica carrier, on which the preparation, in particular a biochemical preparation such as viruses, antigens or the like, is provided. For this purpose, the glass or mica carrier is pretreated with a primer, such that active groups can be fixed thereto for binding the preparation. A glass or, in particular, mica carrier has the advantage of having a particularly smooth carrier surface, which is necessary for obtaining a proper resolution. Indeed, with a microscope, it will have to be possible to observe particularly small elements on the carrier surface.

It is particularly difficult to place such active groups on a glass or mica carrier in a suitable manner. In the first place, the adhesion to such carrier is very difficult to realize, which renders the carriers costly. In the second place, no proper, uniform, densely packed distribution of the active groups can thereby be effected. This means that the resolving power of the microscope is considerably reduced by this preparation carrier, or at least that the resolving power of the microscope cannot be optimally utilized, while it moreover involves a relatively great risk of measuring errors . A further drawback of mica or glass preparation carriers is that they are particularly vulnerable, which complicates treatment thereof and increases the risk of damage and loss.

The object of the invention is to provide a method for manufacturing a preparation carrier of the type described in the preamble, wherein the drawbacks mentioned of the known methods and preparation carriers are avoided, while the advantages thereof are maintained. To that end, a method according to the invention is characterized by the features of claim 1.

Plastic is in principle a favorable material for manufacturing preparation carriers, in that it is relatively simple to process and relatively strong, while a proper binding thereto of different preparations, in particular biochemical preparations such as viruses, antigens, peptides and the like, can be obtained. However, plastic has as a drawback that the surface roughness thereof has hitherto proved to be too high for it to be suitably used as preparation carrier for use with Atomic Force Microscope or a like form-directed examination.

Surprisingly, it has now been found that by a method according to the present invention, a smooth plastic surface can be obtained such that it is in fact suitable, or at least much better suitable, as carrier surface for preparations in such examination. Indeed, by forming the plastic layer, treated thermally or chemically, against a surface of a carrier base with a suitable surface roughness, it appears that the surface roughness of the surface lying against the carrier base can thereby be reduced considerably. Thus, for instance, a reduction of the surface roughness by a factor of 5-20 or more can be realized. This means that elements of a preparation that are bound to the carrier surface can have particularly small dimensions, while the form can nevertheless be optimally determined thereby, for instance with an Atomic Force Microscope, or at least the presence thereof can be established on the basis of at least the form. These elements can for instance have dimensions in the order of magnitude of 20 nm (viruses) , or 3 nm (antibodies) or even smaller, in the order of, for instance, 1 nm.

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

By at least partially melting the plastic against a surface of the carrier base, an optimal distribution of the plastic can be effected in a particularly simple manner. Moreover, in that case, for instance plastic film or sheet can readily be started from. However, it is also possible to cause for instance polymerization of the plastic layer to take place on the carrier surface, or to chemically treat the plastic such that deliquescence against the surface of the carrier base occurs . Without wishing to be bound to any theory, the particular smoothness of the obtained carrier surface seems to result at least partly from the use of a .particularly smooth carrier base and the absence of adhesion to the carrier base. Hence, it seems that a method according to the present invention can be optimized by using a carrier base having an optimal smoothness and the absence of adhesion between the plastic and the carrier base. However, also with 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 features of claim 3.

The use of a plastic having at least one active group for the relevant preparation offers the advantage that no final processing is necessary. A group suitable for the covalent binding of antigens offers the advantage that such preparation carrier is in particular suitable for use in biotechnology.

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

When the plastic used is not directly, or at least not sufficiently suitable for binding the relevant preparation, it is preferred that the carrier surface be treated in such a manner that on, or at least in the carrier surface, one or more active groups for the relevant preparation be provided, again in particular groups for the covalent binding of antigens such as a -COOH group. The advantage thus achieved is that as plastic for the carrier surface, a material can be used having particularly suitable properties therefor, such as, for instance, polyethene, in particular high-molecular polyethene, while the treatment of the carrier surface provides that the binding of the antigens is yet effectively enabled. In this respect, the advantage of plastic over, for instance, mica and glass is that such treatment is possible in a particularly simple and effective manner, while in each case a suitable treatment can be selected, depending on the preparation to be bound.

In further elaboration, such method is preferably characterized by the features of claim 5.

By grafting the carrier surface with a plastic, a carrier surface that in itself binds insufficiently, if at all, can readily be treated for obtaining the desired capacity for binding, in particular covalently binding. Especially the use of acrylic acid or methyl acrylate is particularly suitable therefor.

In a further advantageous embodiment, a method according to the invention is further characterized by the features of claim 8. Surprisingly, it has been found that as the case may be, the surface roughness of a carrier surface can be further reduced by introducing -NH₂ groups in, or at least on the carrier surface. Thus, the surface roughness of a polyethene treated with acrylic acid or methyl acrylate can for instance be reduced thereby such that it can as yet be rendered suitable, or at least better suitable, for use in Atomic Force Microscope examination. In further elaboration, a method according to the invention is further characterized by the features of claim 9, preferably by the features of claims 9 and 10.

By contacting a solution of a suitable monomer with the carrier surface and subsequently treating the plastic and solution, such that polymerization of at least a portion of the monomer occurs, a thin so-called adhesive layer can be provided on the carrier surface in a particularly simple manner, which adhesive layer is properly capable of effecting the desired bindings, in particular covalent bindings. By means of suitable irradiation, this polymerization can be effected and checked in a particularly effective manner. In this respect, it is preferred that the polymerized adhesive layer have a relatively slight thickness, to preserve a proper adhesion and a sufficiently flat surface.

Particularly suitable as carrier base are surfaces formed from, for instance, mica or glass, or materials having comparable surface roughness, hardness and/or porosity. In particular mica proves to be highly suitable therefor, especially because of the substantially atomic flatness thereof .

The invention further relates to a preparation carrier for use in form examination, for instance with an Atomic Force Microscope or the like, characterized by the features of claim 13.

Precisely a preparation carrier having a carrier surface manufactured from plastic, with a surface roughness such that viruses or antibodies or like biochemical elements included thereon are perceptible with an Atomic Force Microscope or a like apparatus, offers the advantage that such preparation carrier is particularly simple to manufacture and adjust to the preparations to be examined, while such preparation carrier can be used in a very simple manner, in particular also because it is relatively strong. The carrier surface being suitable for the covalent binding of the preparation, the advantage achieved is that during use, non-covalently bound and/or non-bound elements of the preparation can readily be washed away or treated otherwise, readily enabling all kinds of assays, known per se, to be performed on the preparation. Precisely the specific binding of elements from the preparation to specific active groups of the carrier surface makes these assays possible.

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

-COOH groups in or at least on the surface readily enable the binding of antigens thereto, through the use of, for instance, soluble carbodiamide (EDC) /sulfo-N-hydroxy- succinimide (sulfo NHS) . -NH₂ groups moreover enable coupling of antigens via, for instance, glutardialdehyde (GDA) . The chemicals used depend on the desired bindings and are directly clear to anyone skilled in the art. Of course, as the case may be, other active groups may also be provided in or on the carrier surface. The invention further relates to the use of an Atomic Force Microscope or like apparatus for biochemical research, characterized by the features of claim 18.

The advantage achieved by the use of such preparation carrier is that an examination with an Atomic Force

Microscope can be performed in a quicker and simpler manner, with a relatively higher resolution, while a large number of antigens can be bound on a particularly small surface. Moreover, the resolving power of the relevant microscope can be utilized in an optimal manner. For that matter, it is observed that in addition to an Atomic Force Microscope, any suitable scanning method can be employed, with or without the use of direct contact, depending on the desired results.

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

To clarify the invention, exemplary embodiments of a method and a preparation carrier will hereinafter be specified with reference to the accompanying drawings. In these drawings:

Fig. 1 shows a carrier base;

Fig. 2 shows a carrier base with a plastic layer applied thereto;

Fig. 3 shows the plastic layer removed 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 on the carrier surface;

Fig. 5 is a schematic representation of a preparation carrier with antigens adhered to the carrier surface; Fig. 5a schematically shows a chemical binding of a

PPV virus to the carrier surface;

Fig. 6 is a much enlarged representation of, respectively, the surface of high-molecular polyethene, the surface of a blown or drawn polyethene film, a carrier surface of polyethene, formed against a carrier base of mica and a surface of glass;

Fig. 7 shows the surfaces of eight carrier surfaces of polyethene formed against mica, treated in different manners with a solution of the relevant monomer and radioactive radiation of a specific intensity; and

Figs . 8a and 8b respectively show a polyethene carrier surface formed against mica, treated with acrylic acid, on which Porcine Parvo Virus and Plum Pox Virus are respectively bound . In this specification, identical or corresponding parts have identical or corresponding reference numerals. Further, as an example in this specification, unless otherwise indicated, a preparation carrier suitable for covalently binding antigens on a carrier surface manufactured from treated polyethene melted against mica, is started from. However, it will be understood that other plastics and another carrier base can be used as well, fqr instance a carrier base of glass and a polypropene, polycarbonate, acrylic acid or methyl acrylate as plastic for the preparation carrier proper. In particular the last-mentioned plastics can offer the advantage that covalent binding thereto is directly possible. Polyethene is relatively inert. However, polyethene offers the advantage of being relatively hard and strong without being brittle. Moreover, other plastics can readily be grafted thereon.

In this specification, in each case a relative flatness measure will be used, the maximal height (Z-axis) of projections above a nominal reference plane being given as percentage of one of the horizontal measures (X-axis) of the scanned surface. In this specification, this horizontal measure is in the order of magnitude of 2-4.5 micrometer. The measure for flatness V is therefore expressed in the following formula:

Z - axis x 100%

X - axis

Examples of the flatness V of materials: mica: V = 0.1%; glass: V = 0.3% (Fig. 6d) ; high-molecular polyethene: V = 10% (Fig. 6a); polyethene film: V = 3% (Fig. 6b) ; and a polyethene face formed according to the invention,

V = 0.6% (Fig. 6c) . These dimensions and values are given only as an example and should not be construed as being limitative in any way. Legend: In the drawing:

D = -COOH

O = -NH₂

Fig. 1 is a sectional side elevation of a carrier base

2, formed from mica, having a top surface 4 with a flatness V of about 0.1%. Hence, this means that on the face 4, there are unevennesses of a maximal height in the Z-direction measured above the nominal face N of at the most a few nanometers, for instance 4-5 nanometer. Hence, the surface 4 of mica is particularly flat. The surface 4 is for instance rectangular, with outer dimensions of 25 x 25 millimeter. The base carrier 2 has a thickness of, for instance, 0.5 millimeter. In the condition shown in Fig. 2, a plastic layer 6 is provided on the smooth top surface 4 of the base carrier 2. In the embodiment shown, this is a polyethene film as shown in Fig. 6b, having an inherent flatness of about 3%. The film layer has a thickness of, for instance, 0.035 millimeter. The film 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 deliquesces on the surfa_.ce 4, after which the whole is cooled. Between the mica base carrier and the plastic layer 6, no adhesion of any significance will occur, allowing the plastic layer 6 to be readily removed from the base carrier 2 again. Surprisingly, it has been found that the surface 8 of the plastic layer 6 that faced the base carrier 2 has obtained a flatness V which is considerably better than the flatness V of the polyethene film used. The flatness of the carrier surface 8 is for instance about 0.6% when no further special measures are taken. It is further observed that, as the case may be, deliquescence of at least the part of the plastic layer 6 facing the base carrier 2 can also be effected, or at least partially effected, by for instance a chemical reaction.

Fig. 3 shows a preparation carrier 1 formed according to the present invention, with the carrier surface 8 facing upwards. In the embodiment shown, for instance polyethene is used as plastic, which is relatively inert. As a result, binding, in particular the covalent binding thereto of biochemical elements is in fact not possible. Fig. 3A shows an alternative embodiment, wherein, as plastic layer 106, a plastic is used containing active groups 112, symbolically represented by spheres placed on rods. Such a plastic can for instance be a polycarbonate, an acrylic acid or methyl acrylate, in which for instance -COOH or -NH₂ groups are present as active groups 112, in the drawing symbolically represented by, respectively, a square and a sphere on a rod. Fig. 4 shows a preparation carrier 1 having a plastic layer 10 grafted thereon, for instance a polymerized layer of acrylic acid or methyl acrylate. Such layer 10 can be applied to the plastic carrier layer 6 of polyethene or another plastic as follows.

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

Suitable monomer solutions are, for instance, a 0.6% or 6% acrylic acid (AC) monomer solution or a 0.6% or 6% methyl acrylate (MA) monomer solution. These solutions can for instance be irradiated with γ-radiation of, for instance, 2 or 12 kilo Gray (kGy) . By a suitable choice of the irradiation time, a desired thickness of the relevant polymerized layer is thereby obtained on and partially in the carrier surface 8. Such layer is preferably as thin as possible, having a thickness of, for instance, a few molecules or chains, so that the flatness of the carrier surface 8 is preserved as much as possible or even further increased.

Figs. 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 appears from Fig. 7, in particular the surfaces shown in Figs. 7c, 7d and 7h are particularly flat and hence extremely suitable for preparation examination. The coding successively gives the carrier plastic (PE) , the concentration of the solution (in %) , the amount of irradiation (in kGy) and the grafting plastic (AC or MA) used. Of course, other combinations are also possible, for instance more or fewer or other monomers, other exposure amounts, other polymerization methods and other carrier plastics. Suitable choices therefrom are directly clear to anyone skilled in the art and can be determined without further invention.

The viruses or antibodies to be bound have or are provided with active groups, for instance -COOH groups and/or -NH₂ groups, which can be coupled directly or via linkers to the active groups 12 on or at least in the carrier surface 8, 10. Thus, for instance -NH₂ groups of a virus can be coupled to a -COOH group or an -NH₂ group of the carrier surface 8, 10, while -COOH groups of a virus can for instance be coupled to -NH₂ groups of the carrier surface 8, 10. As linkers, different chemicals can be used, for instance HMDA

(Hexamethylenediamine) or EDA (Ethylenediamine) . Thereby, for instance -NH₂ groups can be introduced as active groups in or on a carrier surface 8, 10 which only or substantially comprises for instance -COOH groups as active groups 12. HMDA can be used by coupling of Boc HMDA

(Butyloxycarbonylhexamethylenediamine) via DCC (Dicyclohexylcarbodiimine) to the -COOH groups, whereby, after Boc-deprotection, -NH₂ groups become available for the coupling of antigen. When EDA is used, a surface 8, 10 treated with methyl acrylate can subsequently be treated with said Ε1DA for, for instance, 72 hours at 40°C, with active - NH₂ groups becoming available. The first carrier surfaces are for instance PE (0.6/2Ac) -Hmda and PE (0.6/l2Ac) -Hmda, while the second type of surface for instance meets PE(0.6/2MA)- EDA.

The other surfaces shown in Fig. 7 are less flat. Introduction of -NH₂ groups into these surfaces, for instance in the manner described above, surprisingly leads to an improvement of the flatness V of these surfaces. This means that these surfaces, through the introduction of said -NH₂ groups therein, become also or at least even better suitable for use as preparation carrier for at least form-directed examination.

Fig. 5 schematically shows how a number of viruses 14 are coupled to the active groups of a preparation carrier 1. Similarly, coupling to a preparation carrier 101 as shown in Fig. 3A is possible. Fig. 5A schematically shows in what manner chemical binding of a PPV virus to a carrier surface is possible. To the resulting surfaces with -COOH groups, antigens and the like can be coupled via, for instance, EDC (soluble cabodiamide) /sulfo NHS (sulfo-N-hydroxy- succinimide) , known from the Pierce catalogue. To the resulting surfaces with -NH₂ groups, antigens and the like can be coupled via, for instance, glutardialdehyde (GDA) . This last coupling is shown in Figs. 5A and 8 and can 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 a like apparatus, while the presence and/or form of the bound viruses 14 can accurately be determined as a result of the high flatness of the carrier surface 8. From the examples shown in Fig. 8, it for instance appears that 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 as important advantage over the prior art that in a particularly simple manner, different types of active groups can be provided on, or at least in the carrier surface, such as the -COOH groups and -NH₂ groups mentioned. According to the desired application and the desired bindings, the carrier surface can be treated in a suitable manner, if necessary. Moreover, the active groups can be provided so as to be particularly close together, so that a high density of the elements to be detected from the preparation can be obtained, for instance with an occupancy of 10% or more. Occupancies of, for instance, 70-100% are possible. Thus, the resolving power of the detection technique employed can be increased considerably, or at least be utilized in a more optimal manner. The flatness of the carrier surface 8 can possibly be further increased through the use of appropriate techniques, for instance vacuum techniques for placing and melting the plastic layer 6 on the carrier base 2, or at least causing it to deliquesce thereon. This prevents gas inclusions from possibly leading to unevennesses .

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

For instance, other plastics may be used for forming the carrier surface and/or for grafting the layer 10 thereon. Suitable plastics may for instance be selected on the basis of the desired active groups, the desired hardness or flexibility, the desired combination of carrier plastic and grafting plastic, possible resistance to, for instance, chemicals, irradiation, exposure and the like. Such choices will be readily understood by anyone skilled in the art within the framework of the invention. Further, preparation carriers according to the present invention may also be used for other examinations, for instance examinations involving the use of markers for establishing the presence of specific elements, for instance fluorescent, coloring or radiant markers. In the exemplary embodiments shown, the plastic layer is in each case provided on the base carrier, yet it is of course also possible to process a plastic layer with a sufficiently smooth surface of a base carrier that is moved against or along the surface of the plastic layer, for instance a base carrier of mica or glass. It is also possible to cause polymerization of a plastic to take place on a base carrier having the desired smoothness or to effect the formation of plastic having suitable properties thereon in a different manner. Of course, all kinds of different preparations may be bound on a preparation carrier according to the present invention. The viruses described only serve as example. These and many comparable variations are understood to fall within the framework of the invention outlined by the claims .

Claims

1. A method for manufacturing a preparation carrier for use in preparation examination, in particular form-directed examination, such as Atomic Force Microscope (AFM, SFM) examination, wherein: - on at least one surface of a carrier base, a layer of plastic is provided, wherein the plastic layer is treated thermally and/or chemically, such that the surface roughness of the side of the plastic that faces the carrier base is reduced, while it substantially does not adhere to the carrier base, whereupon the plastic is removed from the carrier base, with the released, relatively smooth surface of the plastic forming a carrier surface.

2. A method according to claim 1, wherein the plastic is provided over the at least one relevant face of the carrier base by melting said plastic at least partially.

3. A method according to claim 1 or 2 , wherein as plastic, a monomer or polymer is used having at least one active group for the relevant preparation, in particular a group for the covalent binding of antigens, such as a -COOH group .

4. A method according to claim 1 or 2 , wherein the carrier surface is treated such that the carrier surface comprises at least one active group for the relevant preparation, in particular a group for the covalent binding of antigens, such as a -COOH group.

5. A method according to claim 4, wherein the carrier surface is grafted with a plastic, in particular by means of a monomer or polymer, preferably acrylic acid or methyl acrylate .

6. A method according to claim 5, wherein the carrier surface is treated with acrylic acid or the like and is further treated for obtaining -NH₂ groups thereon, preferably by coupling hexamethylenediamine (Hmda) thereto.

7. A method according to claim 5, wherein the carrier surface is treated with methyl acrylate or the like and is further treated for obtaining -NH₂ groups thereon, preferably by means of ethylenediamine (EDA) .

8. A method according to any one of claims 5-7, wherein by introduction of -NH₂ groups in, or at least on the carrier surface, the surface roughness thereof is reduced.

9. A method according to any one of claims 4-8, wherein at least the plastic layer on at least the carrier surface is brought into contact with a solution of a monomer, whereupon the plastic and the solution are treated such that polymerization of at least a portion of the monomer occurs on the carrier surface, for which purpose, preferably, the plastic together with the solution is exposed to radiation.

10. A method according to claim 9, wherein the carrier surface is provided with a polymerized adhesive layer of a relatively slight thickness, preferably a thickness of at the 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 having a particularly low surface roughness of at least the face to which the plastic is applied, preferably having a surface roughness in the order of magnitude of atomic roughness or slightly thereabove .

12. A method according to claim 11, wherein a base carrier is used of which at least said face is manufactured from mica or glass or a material which is comparable therewith in respect of surface roughness, hardness and porosity, preferably from mica.

13. A preparation carrier, in particular for use in Atomic Force Microscope (AFM, SFM) or like form-directed examination of a preparation, in particular a biochemical preparation, said preparation carrier having a carrier surface manufactured from plastic, wherein the carrier surface has a surface roughness such that viruses or antibodies or like biochemical elements included thereon are perceptible with an Atomic Force Microscope (AFM/SFM) or a like apparatus, wherein the carrier surface is suitable for binding the preparation at least covalently.

14. A preparation carrier according to claim 13, wherein the carrier surface is formed by melting the plastic at least partially on a carrier base having a surface roughness less than or approximately equal to the surface roughness of the carrier surface .

15. A preparation carrier according to claim 13 or 14, wherein the plastic is a polymer, in particular polyethene or polypropene .

16. A preparation carrier according to any one of claims 13-15, wherein the carrier surface is grafted by means of a monomer or polymer, preferably acrylic acid or methyl acrylate .

17. A preparation carrier according to any one of claims 13-16, wherein the carrier surface comprises at least -COOH groups and preferably -COOH 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 bound to the carrier surface.