Recognizable carrier for optical measurement methods

Abstract

The invention relates to a recognizable carrier for determining physical, chemical or biochemical interactions by means of optical measurement methods. The carrier comprises a surface that defines a substrate surface and that has a base layer coated with reactive elements, which are bonded to receptor molecules, wherein the base layer and/or the reactive elements are provided with a pattern of holes which forms a code and/or the reactive elements are provided with linker molecules or markers which form a code. The substrate surface may additionally have a macroscopically planar pattern which is applied using laser light or chemical etching and forms a code. The invention likewise relates to a method for producing a recognizable carrier for spectroscopic processes and/or intensiometric tests to determine said interactions. The code to recognize the carrier can be controlled via a read-out unit coupled to the photometric analysis unit. Such a carrier can be used to analyze biomolecules during security checks, access controls or in-vitro diagnostics.

Claims

1. A recognizable carrier for the determination of physical, chemical or biochemical interactions using optical measurement methods, comprising: a plate surface, a well formed in the plate surface, the well having a sidewall and a bottom surface which is at least partially chemically activated and defines a substrate surface, a base layer disposed on the substrate surface and coated with reactive elements that binds to receptor molecules, and an implicit coding for controlling authenticity and quality of the carrier, wherein the implicit coding is formed by a void pattern and a plurality of spots formed on at least one of the base layer and the reactive elements, wherein the void pattern is formed by a plurality of voids or holes representing the implicit coding, and the plurality of spots are disposed in a predetermined arrangement and have different heights representing the implicit coding.

2. The carrier of claim 1, wherein the carrier is planar.

3. The carrier of claim 1, wherein the substrate surface comprises an additional microscopic two-dimensional pattern applied with laser light or by chemical etching to form a coding forming pattern, and wherein the void pattern of the base layer and at least partially the pattern of the reactive elements correspond to the macroscopic two-dimensional pattern of the substrate surface.

4. The carrier of claim 1, wherein the reactive elements comprise basic structures derived from alkenes with oligomer molecular components.

5. The carrier of claim 1, wherein the carrier is made of glass in form of a flat carrier or a micro-titer plate.

6. The carrier of claim 1, wherein the carrier is made of plastic in form of a flat carrier or a micro-titer plate.

7. The carrier of claim 5, wherein the glass forming the micro-titer plate comprises borosilicate.

8. The carrier of claims 1, wherein the plate surface of the carrier is reflecting or comprises elements selected from aluminum, zinc oxide, titanium oxide or mixtures thereof.

9. The carrier of claim 1, wherein the void pattern is formed on both the base layer and the reactive elements.

10. The carrier of claim 1, wherein the sidewall of the well has a predetermined height, and the heights of the plurality of spots are lower than the predetermined height of the sidewall of the well.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) The invention will now be described in more detail with reference to exemplary embodiments and the appended drawings.

(2) It is shown in:

(3) FIG. 1a a micro-titer plate with a plurality of wells arranged in rows, with a partially applied coding;

(4) FIG. 1b a top view onto a selected well of the micro-titer plate in the region of the cross-sectional line I-I in FIG. 1;

(5) FIG. 1c a cross-section through the micro-titer plate having wells with a coating along the cross-sectional line I-I of FIG. 1; and

(6) FIG. 1d a cross-section through a selected well of the micro-titer plate, as marked in FIG. 1c, with a coding in form of reactive elements applied as spots and arranged in different geometries.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(7) Exemplary Embodiments

(8) When a recognizable carrier modified according to the invention is to be produced, the process for producing a surface coating can generally be described by the steps cleaning and activation of the substrate surface, forming a base layer, preferably by silanization, immobilization of the reactive elements in form of a selected biopolymer, and functionalizing the immobilized biopolymer with so-called receptor molecules to be defined in more detail below.

(9) Coating a Glass Carrier with a Macroscopic Two-dimensional Pattern Forming a Coding

(10) I.1 Forming a Coding on the Substrate Surface by Spotting

(11) According to this exemplary embodiment, an object slide carrier is used as an exemplary glass carrier. The use of this object slide carrier is only one example. It is also possible and, depending on the application, even preferable, to use a glass known to a person skilled in the art that is different from the object slide carrier, for example a glass with an optical coating.

(12) The glass carrier is initially cleaned by immersion for at most 1 minute in 6 N KOH and subsequently rinsed with ultrapure water. The glass carrier is then activated by generating OH groups on the glass surface. This glass surface will hereinafter be referred to as substrate surface.

(13) The substrate surface is activated with freshly prepared piranha solution. With full-surface activation, the glass carrier is treated for about 15 minutes with the freshly prepared piranha solution in an ultrasound bath, and subsequently thoroughly rinsed with ultrapure water and dried in a flow of nitrogen. The term piranha solution refers to peroxymonosulfuric acid which is prepared in the present example by addition of concentrated sulfuric acid to a 30% hydrogen peroxide solution in a volume ratio of concentrated sulfuric acid to hydrogen peroxide solution of 3:2.

(14) It proved initially quite difficult to form a recognizable reliable coding on the substrate surface during this activation. The problem is here to intentionally apply a precisely defined, spatially resolved pattern. This was attained in one embodiment by spotting, using the TopSpot method. With this method, a contactless and especially simultaneous and spatially well resolved application of the piranha solution on the substrate surface in a dense pattern corresponding to the desired coding pattern became possible.

(15) The TopSpot technology is commercially available from the company Biofluidix GmbH, Freiburg, Germany.

(16) The TopSpot method is based on a micro-structured print head made from a silicon carrier with an array of up to 96 circular nozzles. The silicon core is surrounded by Pyrex. The piranha solution is then filled into a reservoir of the print head provided and is transported exclusively by capillary forces to the centrally located nozzles which are arranged in a grid pattern of 500 m. The print head is then installed in a print module which includes the actual drive. A piston, which is movably supported on a flexible seal above the nozzle window, is used for releasing droplets. The piston is displaced by controlling a piezo-actuator. This displacement produces a pressure pulse in the closed air chamber between the piston and the nozzles. This pressure pulse operates on all nozzles simultaneously. As a result, a single liquid droplet is simultaneously released from each of the nozzles. The volume of these droplets is in this pneumatic method about 1 nL (de Heij, B., Daub, M., Gutmann, O., Niekrawietz, R., Sandmaier, H. & Zengerle, R., (2004) Highly parallel dispensing of chemical and biological reagents, Analytical and Bioanalytical Chemistry, 378, 119-122).

(17) With this method, a precisely defined, spatially resolved pattern can be reproducibly produced on the substrate surface, making it possible to distinguish regions defined by the piranha solution from the non-activated regions. With the same success, i.e., with the same quality, patterns with different spatial resolution were formed on the substrate surface.

(18) I.2 Forming a Coding on the Substrate Surface by Printing

(19) In this exemplary embodiment, an object slide carrier was again used and activated with piranha solution in the same manner as described in the first exemplary embodiment under I.1.

(20) A recognizable reliable coding on the substrate surface through specific application of a precisely defined, spatially resolved pattern was formed in this exemplary embodiment by printing using a MicroDrop dosing system.

(21) Such MicroDrop dosing system is commercially available from microdrop Technologies GmbH, Norderstedt, Germany.

(22) The MicroDrop dosing system is based on the technology of inkjet printers. With a piezo-pump consisting of a capillary with a piezoelectric element, a very rapid pressure increase is generated which propagates with the velocity of sound through the fluid to the nozzle opening and actively displaces a quantity of fluid in a capillary, causing a fine fluid column to exit the nozzle with high acceleration. The ejection is so fast that the fluid is ejected without wetting the capillary edge. Droplets in a range of 50 pl to 500 pl, corresponding to a droplet diameter of 300 m to 100 m, can be produced. Either a XYZ table or a suitable robot is used for positioning the droplets (Schober, A., Guenther, R., Schwienhorst, A., Doering, M. Lindemann, B. F., (1993), Accurate high-speed liquid handling of very small biological samples, Biotechniques, 15, 324-329).

(23) This method also produced a precisely defined, spatially resolved pattern on the substrate surface, which made it possible to accurately distinguish the regions activated by the piranha solution from the non-activated regions.

(24) With the same success, i.e., with the same quality, patterns with different spatial resolution were formed on the substrate surface.

(25) I3. Formation of a Coding on the Substrate Surface with O.sub.2 Plasma

(26) As an alternative to the methods described under I.1. and I.2. for generating precisely defined, spatially resolved patterns on the substrate surface by specific activation with piranha solution, activation can also be performed with O.sub.2 plasma.

(27) Preferably, glass carriers with a coating that is unstable with respect to acid are used. Such glass carriers are known in the art. Possible coatings include, for example, TiO.sub.2 and ZnO.

(28) However, carriers made of plastic can also be used, such as carriers made of TOPAS (cyclo olefin copolymer (COC) from the company Topas Advanced Polymers GmbH, Frankfurt a.M., Germany) or PMMA (polymethylmethacrylate), to mention only an exemplary selection.

(29) In the exemplary embodiment, a TOPAS plastic carrier was activated for 15 minutes in an oxygen plasma (300 W, 0.8 mbar O.sub.2). Advantageously, the surface of this treatment is simultaneously also cleaned, potentially eliminating a prior cleaning step.

(30) In order to reproducibly produce a precisely defined, spatially resolved pattern on the substrate surface with this kind of activation, a mask made of a Teflon corresponding to the respective desired pattern was used. This mask was pressed onto the substrate surface during the activation. All parts of substrate surface covered by the Teflon mask remained non-activated.

(31) This method was then also capable to reproducibly produce a precisely defined, spatially resolved pattern on the substrate surface, which made it possible to accurately distinguish regions activated by the oxygen plasma from the non-activated regions covered by the mask.

(32) With the same success, i.e., with the same quality, patterns with different spatial resolution were formed on the substrate surface.

(33) As an alternative to the activation by oxygen plasma, activation with a barrier discharge can also be used.

(34) II. Forming a Coding in the Base Layer

(35) II.1. Forming a Coding in the Base Layer with a Full-area Activated Substrate Surface

(36) A glass carrier is cleaned and activated, as described initially in general terms above in the first and second paragraph of I.1.

(37) The substrate surface of the glass carrier is then prepared for a surface treatment in form of silanization with epoxy groups, which in the exemplary embodiment is performed with epoxy silane in form of 3-(glycidyloxypropyle) trimethoxy silane (GOPTS). As an alternative, for example, phenethyl trichloro silane, octadecyl trichloro silane or other corresponding compounds can be used.

(38) 15 L/cm.sup.2 GOPTS are applied on the cleaned and activated substrate surface of the glass carrier.

(39) II.1.1. Forming a Coding by Spotting

(40) In this case, the TopSpot method described in detail above under I.1. is used again. Different patterns were applied. It was observed that with this method the GOPTS could be applied each time on the substrate surface contactless and more particularly simultaneously and with clean spatial resolution, and in addition with a tight grid pattern corresponding to the desired coding pattern.

(41) II.1.2. Forming a Coding by Printing

(42) Again, the MicroDrop dosing system was used, which was described above in detail under I.2. This method was also capable of reproducibly producing a precisely defined, spatially resolved pattern on the substrate surface, wherein regions covered by GOPTS could be clearly differentiated from the regions that were not covered.

(43) With the same success, i.e., with the same quality, patterns with different spatial resolution were formed on the substrate surface.

(44) II.1.3. Forming a Coding Through Deactivation with Laser Light or Chemically

(45) A glass carrier is cleaned and activated, as initially described in general terms above in the first and second paragraph of I.1.

(46) The entire area of the substrate surface of the glass carrier is then subjected to a surface treatment in form of a silanization with GOPTS.

(47) Subsequently, precisely defined regions are deactivated with laser light with spatial resolution. In the exemplary embodiment, a conventional IR laser is used.

(48) Precisely defined regions can also be chemically deactivated with spatial resolution. The silanol groups are here condensed with precise definition.

(49) II.1.4. Forming a Coding by Using Different Silanes

(50) In accordance with the aforedescribed method of spotting with to the TopSpot method or printing with the MicroDrop dosing system, the base layer is formed from two different silanes, on one hand GOPTS and on the other hand a silane lacking reactive groups in form of the epoxy groups.

(51) Spotting and/or printing are then specifically performed such that regions of the substrate surface to be activated are covered with GOPTS, whereas the other regions are covered according to a defined pattern with the silane that lacks epoxy groups and therefore does not enable activation of the base layer at these locations. In this way, the code is formed in the base layer.

(52) II.1.5. Forming a Coding by Subsequent Deactivation

(53) The carriers treated over the surface area with GOPTS can also be rendered inert for subsequent binding of the reactive elements by spatially-resolved deactivation of the active group in the step to be described below. The deactivation can also be accomplished, for example, through specific application of water, wherein the deactivation takes place by hydrolysis. The specific application can once more be performed with the TopSpot method or with the MicroDrop dosing system.

(54) II.2. Forming a Coding in the Base Layer Through Activation of the Substrate Surface With Coding

(55) A cleaned and activated glass carrier is used, as described above under I. The substrate surface has already been provided with a coding according to one of the methods described above under I.

(56) The substrate surface of the glass carrier is thereby prepared for a surface treatment in form of silanization with epoxy groups, which in this exemplary embodiment is performed again with an epoxy silane in form of 3-(glycidyloxypropyle) trimethoxy silane (GOPTS). As an alternative, phenethyl trichloro silane, octadecyl trichloro silane or other corresponding compounds can again be used.

(57) 15 L/cm.sup.2 GOPTS are applied to the cleaned and activated substrate surface of the glass carrier. However, these can bind to the substrate surface only where the substrate surface is also activated. In this way, the code already formed on the substrate surface can be accurately imaged and reproduced in the base layer in every detail. Silane that is not bonded to the substrate surface is simply washed out by rinsing.

(58) II.3 Further Treatment of the Base Layer that was Activated and Coded Through Silanization

(59) After GOPTS or alternatively another silane has been applied to the substrate surface, the carrier treated in this manner is, after an incubation time of one hour in a dry empty gas chamber, briefly rinsed with dry acetone having a water content of 0.1%, particularly monitoring for any GOPTS residues.

(60) Because of the humidity of the air, the carrier(s) must be quickly further processed.

(61) II.3.1 Coating with the Biopolymer Aminodextran as Reactive Element

(62) Subsequently, aminodextran (AMD) is applied to the base layer prepared in this manner by applying 15-20 L/cm.sup.2 aminodextran solution (corresponding to 10 mg AMD in 20 L ultrapure water).

(63) Application can hereby also occur by spotting or printing in the aforedescribed manner.

(64) After incubation overnight in a chamber saturated with water vapor, the sample is thoroughly rinsed with ultrapure water and dried in a nitrogen flow.

(65) II.3.2 Coating with the Biopolymer Di-amino-polyethylene Glycol as Reactive Element

(66) For immobilizing di-amino-polyethylene glycol (DA-PEG), 20 L/cm.sup.2 DA-PEG solution (corresponding to 4 mg DA-PEG in 1 mL dichloromethane) are applied to a glass carrier silanized with GOPTS and incubated in an open tray at 70 C., preferably overnight. Excess DA-PEG is subsequently rinsed with ultrapure water and the carrier is subsequently dried in a nitrogen flow.

(67) The free COOH end groups of the PEG are then activated to an active ester.

(68) III.3.3. Forming a Coding in the Coating Formed by Further Reaction with the Biopolymer

(69) In an analog manner, the biopolymer can only bind to the silane groups of the base layer where corresponding epoxy groups exist. The biopolymer therefore again accurately reproduces the code defined by the silane groups of the base layer in every detail.

(70) So as not to unnecessarily increase the number of examples, reference is made in general to the resulting possibilities for coding which have each been tested in experiments:

(71) When a coding was already performed on the substrate surface, as described above, the formed code is accurately imaged in the base layer in every detail, i.e., accurately reproduced by the silane groups. The biopolymer coupled to the epoxy groups of silane is also accurately reproduced in every detail. As a result, a very clean traceable coding is reproduced in every layer with almost zero error rate. This is important for the safety, especially in applications in the medical field.

(72) The possibilities proposed so far also demonstrate that the coding can first be provided in the base layerdisregarding the substrate surface. The coding can also be implemented in the region of the coating with the reactive elements in form of the biopolymers, without necessarily requiring prior measures for coding the substrate surface and/or the base layer with respect to the silanization. This will be described in more detail in the following exemplary embodiment.

(73) III.3.3. Forming a Coding with the Biopolymer

(74) The reaction with PEG following the silanization with GOPTS is performed by using DA-PEG composed of two different PEG species. This is to be understood that a short PEG with only one head group is used in excess together with a longer PEG with two head groups.

(75) DA-PEG with a molecular weight of about 2000 DA was used as a short PEG with only a single head group and DA-PEG with a molecular weight of about 6000 DA was used as a longer PEG with two head groups. The different PEG species were applied again on the carrier that was prepared by silanization by spotting or printing according to the aforedescribed methods in a specific predetermined pattern commensurate with the desired coding.

(76) In addition, experiments were also performed by using a carrier where the silanized base layer already included the pattern of the desired coding and only the biopolymer needs to react. DA-PEG from two different PEG species was here also used, as described above. The PEG species were mixed, providing the advantage of producing a surface with less reactive groups, while however simultaneously providing improved binding conditions for the additional binding with the receptor molecules, for example in form of antibodies.

(77) II.4 Immobilization of the Receptor Molecules

(78) The aforedescribed coating with the reactive elements, which had on its surface or was coated on its surface with either the coding transferred using at least one of the above treatment steps and/or a new coding produced by specific application of DA-PEG having different molecular weights, can in the following be used for immobilizing receptor molecules.

(79) Alternatively, a further treatment is performed by re-functionalization such that they subsequently have carbonyl functions.

(80) For this purpose, the amines forming the coating of the reactive elements are incubated with 15 L/cm2 glutaric acid anhydride solution (corresponding to 2 mg glutaric acid anhydride in 1 mL dry DMF) for six hours in a glass chamber saturated with DMF and subsequently rinsed with DMF and ultrapure water. The carrier is then dried in a nitrogen flow.

(81) A coding applied to the respective amine, as described above in one of the alternative approaches, is transferred substantially error-free.

(82) II.4.1 Immobilization of the Receptor Molecules with Reactive Elements which Each have at Least One Carbonyl Group

(83) For immobilizing a receptor molecule with a carbonyl group, an amino-functionalized glass carrier of the type described above is used. A solution of 1 mg receptor molecule in 10 L DMF with a water content of 0.1% and 50 mL DIC (di-isopropyl carbodiimide) is applied to the coded base layer with the biopolymer and incubated for at least six hours in a chamber saturated with DMF.

(84) For immobilizing receptor molecules which each have an amino group, the biopolymers re-functionalized, as described above, so that each have a carbonyl function, are activated with 15 L/cm.sup.2 of a NHS/DIC solution (15 mg NHS in 100 L dry DMF and 30 L DIC) for about four hours in a chamber saturated with DMF, whereafter rinsed with dry DMF and dry acetone, and dried in a nitrogen flow.

(85) The receptor molecules can subsequently also applied to the surface prepared in this manner from aqueous solutions and with different concentrations.

(86) The coding transferred from the substrate surface and/or the silanization of the base layer and/or the coating of the base layer with the biopolymer (amine) can still be transferred to the receptor molecules essentially error-free because the corresponding non-activated regions of the biopolymers are incapable of binding receptor molecules. It is also possible to apply a coding for the first time by specifically applying the receptor molecules commensurate with a desired pattern.

(87) In the experiments performed for the purpose of the present invention, these patterns are applied by spotting as well as by printing according to the aforedescribed methods.

(88) Antibodies which are bonded covalently via peptide bonds to the coating formed by the biopolymers should be mentioned as an example of receptor molecules used for demonstration purposes in the described exemplary embodiments, which are also of great importance for practical applications.

(89) IV. Coating a Micro-titer Plate Made of Glass

(90) For sake of simplification, it is noted at this point that the aforedescribed experiments and coding steps were not only tested on an object carrier, but also in another series of experiments on a micro-titer plate made of glass.

(91) A conventional commercially available rectangular micro-titer plate, also referred to as well-plate or multi-well plate, with an average surface roughness was used, which has 8 rows and 12 columns of isolated cavities, also referred to as troughs or wells. The fill volume of the cavities is between 0.3 and 2 mL.

(92) In addition to these 96-type micro-titer plates, a micro-titer plate with only 24 cavities arranged in 4 rows and 8 columns was used, with the cavities having each a fill volume of 0.5 to 3 mL.

(93) The wells of the employed plates have a flat bottom or a bottom with a U-shaped depression. Each of the bottoms or each of the depressions itself forms a surface which defines, in analogy to the glass carrier, a substrate surface and which can accordingly have a coding on the substrate surface and/or as a result of the silanization of the base layer and/or the application of a biopolymer on the base layer, which is transferred substantially error-free to the reactive elements.

(94) In the same way, flat carriers and micro-titer plates made of plastic were tested which are also suitable. Commercially available, substantially transparent micro-titer plates were used, which are commercially manufactured from, for example, polystyrene.

(95) In the following, the formation of the coding will again be illustrated with respect to a commercially available glass micro-titer plate with reference to the figures of the drawings. The methods described in detail above for the object carrier made of glass are used for forming the coding(s).

(96) FIG. 1a shows a micro-titer plate 1 having wells 3 arranged in a plurality of rows and columns, to which partially a coding is applied. The micro-titer plate 1 provided with the coding of the invention furthermore includes a conventional plate coding 5 in form of a barcode. The bottoms of the wells 3 have a layer sequence according to one of the aforedescribed embodiments. A well 3 is selected in the region of the cross-sectional line I-I and is illustrated in FIG. 1b in more detail in a top view, with the coating 7 clearly visible. The coating 7 has voids or holes 9 and regions applied by spotting, which will be referred to hereinafter as spots 11. The spots 11 each have a different height. This is shown in FIGS. 1c and 1d.

(97) FIG. 1c shows a cross-section through the micro-titer plate 1 along the line I-I, showing the coating 7 in the wells 3 of the micro-titer plate 1 with a coding in form of layer thicknesses having different heights of the reactive elements applied as spots 11. FIG. 1d shows in more detail the well 3 marked in FIG. 1c, which as a cross-section through the well 3 corresponds to the top view on the same well 3 of FIG. 1b and shows the applied coding in form of spots 11 having different heights of the applied reactive elements.

(98) The identification of a forgery-proof multiple-coding is now available for recognizing the micro-titer plate 1 as a genuine product. This multiple-coding consists of the arrangement of the voids or holes 9, the height of the spots 11. The height of the coating 13 can also be measured independently. The plate coding 5 in form of the bar code is additionally matched in a conventional manner.

(99) The present exemplary embodiments are intended as a detailed description of the carriers according to the invention and their potential use. They are intended to be explanatory and not limiting. Based on the examples and the general description, a person skilled in the art will recognize the large number of additional possible combinations of the codings according to the invention with each other.