EP1242177A1

EP1242177A1 - Method and device for the mask-free production of biopolymers by means of a light diode array

Info

Publication number: EP1242177A1
Application number: EP00991273A
Authority: EP
Inventors: Heinz Eipel; Markus Beier
Original assignee: Deutsches Krebsforschungszentrum DKFZ; BASF SE
Current assignee: Deutsches Krebsforschungszentrum DKFZ; BASF SE
Priority date: 1999-12-23
Filing date: 2000-12-27
Publication date: 2002-09-25
Also published as: AU3164701A; NO20023002L; DE19962803A1; WO2001047627A1; CN1413126A; IL150178A0; US20040026229A1; CA2396721A1; NO20023002D0; JP2003519779A; MXPA02006195A

Abstract

The invention relates to a method and a device for the light-controlled synthesis of biopolymers on surfaces. Patterns of individual sequences (20) are produced on the surfaces by representing an arrangement (1) of electrically and individually controllable light diodes (2).

Description

METHOD AND DEVICE FOR MASK-FREE PRODUCTION OF BIOPOLYMERS WITH A LUMINOUS DIODE ARRAY

The invention relates to a method and an apparatus for the mask-free production of biopolymers, which are synthesized, for example on a slide, by an exposure sequence.

Until now, mask sets have been required for light-controlled DNA chip synthesis for each individual chip. From US 5,412,087 is an area

15 addressable immobilization of oligonucleotides and other chemical polymers on surfaces is known. According to this method, it is proposed that substrates with surfaces that contain components with thiol groups and that have photoactively removable, protective groups can be used to generate fields of immobilized anti-ligands,

20 such as oligonucleotides or other biopolymers. The fields are used to detect the presence of complementary nucleic acids in a liquid sample. The regionally addressable irradiation of predefined regions on the surface allows the immobilization of oligonucleotides and other biopolymers in the activated regions of the

25 surface. Irradiation cycles on different surface areas of the surface and immobilization of different anti-ligands allow the formation of an immobilized matrix of anti-ligands at specific locations on the surface. The immobilization matrix of the anti-ligands allows a liquid sample to be searched for ligands at the same time

30 have a very high affinity for certain anti-ligands in the matrix. In the method proposed here, the surface of the substrate body is irradiated through a mask with a light source which emits a wavelength in the range between 280 and 420 nm, it being possible to select only certain preselectable regions for irradiation with each individual mask.

US 5,744,305 relates to materials applied in the form of a field to a carrier. These serve a synthesis strategy for the production of chemically divergent substrates. Photoactive protective groups of molecules are used to achieve chemical synthesis processes that run in parallel in areas controlled by light. Binary masking techniques are used in the context of an exemplary embodiment. In the disclosed method, various chemical components are synthesized in parallel using a masked radiation source or using an activator. The lighting pattern determines which areas of the slide are prepared for a chemical reaction. The masking technique is also used here to select different areas to be exposed on the slide.

US 5,143,854 relates to a method for the photolithographic synthesis of polypeptides and a search method. In this method, polypeptide fields are synthesized on a substrate by applying photoactive groups to the surface of a substrate which expose certain regions of the substrate to the light in order to activate the regions. An amino acid monomer with a photoactive group is applied to the regions activated in this way, the activation and attachment steps being repeated until polypeptides of the desired length and sequence are synthesized. The resulting field can be used to select those peptides that are able to bind a receptor. In addition to the masking method already mentioned, US Pat. No. 5,143,854 proposes using a diode light source for exposure and exposing the substrate to be exposed in accordance with the partial areas to be exposed. This procedure requires a complex mechanical control mechanism in order to align the substrate exactly in accordance with the areas to be exposed by the light-emitting diode. This mechanical alignment must be carried out each time anew for each new field to be exposed.

The control mechanism places very high demands on the manufacturing accuracy in order to achieve the above-mentioned positioning positions.

In addition to masking the biopolymer regions to be exposed on the slide, it is known from WO 99/42813 to expose DNA sequences or polypeptides or the like by means of a device with controllable micromirrors, the micromirrors forming a coherent field which is composed of electronically addressable individual micromirrors is. A common light source is assigned to this. The biopolymers on the slide are activated in certain patterns, the sequentially offered monomeric building blocks being coupled to the controlled areas. This process continues until all elements of a two-dimensional field on the substrate have reacted with the desired monomer. The micromirror field can e.g. can be controlled in connection with a DNA synthesizer in such a way that the image sequence through the micromirror field is coordinated with the liquid sample applied to the slide.

In the masking method shown, photoactive monomeric building blocks or photoactive surfaces are used to create a site-oriented Enable synthesis. The action of light serves to remove the photoactive groups of the monomeric building blocks or surfaces, so that a synthesis or an immobilization step can then take place at these points of the action of light. In order to bring the light exclusively to the location required for the respective synthesis or immobilization step, masks are used or micromirror fields are controlled.

If, for example in the case of light-controlled oligonucleotide synthesis, n-nucleotides from an ensemble of four different bases are linked in an n-mer sequence, 4 x n masks are required. If a light-controlled synthesis of peptides with a sequence length of n and an ensemble of 20 amino acids is to be carried out, 20 x n masks are required. Such a mask set must not only be provided in advance, but must also be adjusted very precisely during the exposure. This is accompanied by a considerable technical outlay, so that the masking process is not worthwhile for small series, but a new mask set has to be provided for each new synthesis.

The modular light source disclosed in US Pat. No. 5,143,854 allows the slide to be translationally displaceable with corresponding mechanical outlay. Another disadvantage is the fact that the exposure can only take place sequentially and not in parallel.

The invention has for its object to provide a method for the synthesis of biopolymers, which simplifies exposure and activation of individual areas of a biopolymer-containing slide and makes it mask-free. According to the invention, this object is achieved by the features of claims 1 and 15.

The advantages associated with the solution proposed according to the invention are diverse in nature. With very simple means, arrays of

Biopolymers such as Synthesize oligonucleotides and peptides, for example. Furthermore, biopolymers can be immobilized in a light-controlled manner; no masks are needed, the preparation and preparation of which are used

Work steps can be completely omitted. With the removal of the masks, the associated adjustment processes can also be completely dispensed with. There are no complicated and expensive mechanical sliding tables

Control of the individual synthesis sites is still complex

Positioning equipment needed. The exposure steps can all take place in parallel. Furthermore, by eliminating the masks by means of the method proposed according to the invention, the synthesis of individual or small series can also be carried out extremely economically.

In an advantageous embodiment of the method according to the invention, nucleotides and peptides can be synthesized on surfaces. In addition, biopolymers can also be immobilized on the surfaces. Biopolymers are understood to mean, for example, nucleic acids, their analogs (e.g. PNA, LNA), amino acids, peptides, proteins, carbohydrates, and combinations of these. The switching on and off of the light-emitting diodes of the light-emitting diode array from 9, 16, 25 or up to 100 or even more light-emitting diodes is preferably controlled automatically by a computing unit. The computing unit contains the radiation arrangements desired in each case in a stored form.

The exposure time during which the individual light-emitting diodes selected areas of a slide can also be entered via the computing unit irradiate. Such light-emitting diodes are preferably used which emit high-energy radiation in the UV range. In order to shorten the synthesis process by shortening the activation or immobilization times, exposure processes can be carried out simultaneously by the light-emitting diode array containing several light-emitting diodes. A sequential sequence of exposure processes can also be set up.

The simultaneity of the activation of a plurality of light-emitting diodes of the light-emitting diode array can be achieved, for example, by the activation of the light-emitting diodes via the parallel interface of a computer. The parallel execution of exposure cycles, taking into account the sequence of individual exposure times stored in the computing unit, considerably shortens the synthesis of biopolymers. The substrate for the biopolymers to be synthesized thereon is located in a feed device below a translucent area. The feed device can be configured, for example, as a flow chamber in which the chemicals required for the synthesis to be carried out can be offered sequentially. The respective sequences for the individual fields of the array to be synthesized are first entered into the controlling computer. With a corresponding program, the computer controls the individual light-emitting diodes in the light-emitting diode array correlated to sequential and cyclical supply of the individual monomers in accordance with these specifications.

The exposure preferably takes place spatially separate from the chemical synthesis in order to exclude any external disturbing influences during the exposure. In addition to the pro-sequence of biopolymers to be synthesized individually, the sequential sequence of immobilization sites for freely selectable biopolymers can also be stored in the computing unit.

By means of the device proposed according to the invention, parallel, light-controlled synthesis or immobilization of biopolymers can be achieved by computer-assisted parallel control of individual light-emitting diodes without the need for masks. The light-emitting diodes are preferably designed as light-emitting diodes which emit light in the UV wavelength range. For the geometric scaling of the biopolymer arrays, for example, the light-emitting diode array can be optically imaged on the desired scale. Appropriate optical devices are used for this.

The invention is explained in more detail below with the aid of the drawing:

It shows:

Fig. 1 shows a field of, for example, 4 x 4 individually controllable

LEDs with the electrical control lines,

2 shows a fluorescence image of an oligonucleotide array, synthesized using a 4 × 4 light-emitting diode array after hybridization, Fig. 3 four surface fluorescence images in four different

Sensitivity levels to determine a sufficient exposure time with a 3 x 3 LED array,

Fig. 4 shows the structure of a sequence on a chip surface using a

2 x 2 LED arrays.

1 shows the top view of a field with, for example, 4 x 4 individually controllable light-emitting diodes and the associated control electronics.

1 shows a light-emitting diode array 1, which is located below a slide 12 or below a chip surface 19. The arrangement shown in FIG. 1 is a light-emitting diode arrangement 1, which contains 16 individually electrically controllable light-emitting diodes 2; of the light emitting diodes 2 shown, the light emitting diodes A _1? B ₃ and D ₄ are shown in more detail, which are to be controlled for example for one of sixteen DNA sequences.

Each activation of a light-emitting diode 2 of the light-emitting diode array 1 takes place via separate control lines 4, the individual light-emitting diodes 2 being connected to the supply part 8. Furthermore, the individual light-emitting diodes 2 of the light-emitting diode array 1 are each connected to resistors 5, from which further lines extend to memory cells 6 and 7, respectively. The memory cells 6 and 7, in turn, are controlled via a parallel interface 10 provided on a computing unit 22. In the computer 22 shown here only schematically with its parallel interface 10, the DNA sequences 20 and the exposure times required to remove the individual photolabile protective groups or the sequential sequence of immobilization sites can be stored in various files. Furthermore, the computing unit 22 can provide those chemicals required for the synthesis of biopolymers such as, for example, oligonucleotides or peptides, which, depending on the sequence to be treated, react at precisely predefined exposure locations after the unstable photo protection groups have been removed there. Due to the correlation of the sequences, with the inflow of chemicals and the associated exposure locations, a simultaneous exposure of several exposure locations can take place by using the parallel port 10 on the computing unit 22.

The light-controlled synthesis takes place, for example, in a feed device, which can be designed, for example, as a flow cell. The flow of the chemicals necessary for synthesis flowing through the flow cell is controlled by a DNA synthesizer, for example by the computing unit 22. This contains, for example, the DNA sequences 20 in files in stored digital form.

In order to define, within a synthesis cycle taking place in a flow chamber, at which position which of the four nucleotide building blocks - deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine - is to be condensed, photolabile protective groups must be removed on the substrate support at predetermined times. The removal of the photolabile protective groups is necessary because only after their removal can a synthesis and the construction of a DNA oligomer be achieved. The light-emitting diode arrangement 1 according to FIG. 1 exposes the substrate carrier at the locations at which the photolabile protective groups are to be removed individual light-emitting diodes 2 of the light-emitting diode arrangement 1 are preferably designed as individually electrically controllable light-emitting diodes 2. These emit very high-energy radiation, preferably in the ultraviolet range with a wavelength of preferably 360 nm. However, light-emitting diode arrangements 1 can also be used, in which individual light-emitting diodes 2 are received, which emit radiation of a different wavelength, which is different from the UV range. The optimal wavelength of the light-emitting diodes 2 must be matched to the photochemistry used.

By controlling the individual light-emitting diodes 2 of the light-emitting diode arrangement 1, it is determined at which point on the substrate support photo-protection groups are split off in order to enable the attachment of nucleotide building blocks to be coupled. For this purpose, three light-emitting diodes 2 are identified by way of example in FIG. 1, which are denoted by Aj, B ₃ and D. The emitted high-energy radiation from the light-emitting diodes 2, denoted Ai, B ₃ and D ₄ , preferably strikes the locations of the substrate carrier of the feed device and causes the unstable photo-protection groups to be removed at the precisely defined locations. Depending on the substrate applied to the carrier, the exposure time can be different, also depending on the sequence to be generated. The different exposure times that are to be observed by the light-emitting diodes with regard to their on time can also be stored in a file of the computing unit 22 and can thus be incorporated into the proposed exposure method.

By activating the light-emitting diodes 2 corresponding to positions Ai, B ₃ and D ₄ , the protective groups are now removed at these positions on the substrate carrier, so that a chain extension can be achieved only at these well-defined locations on the substrate carrier within this synthesis step. In a subsequent synthesis step, for example, the photolabile protective groups on the substrate can be split off, for example on the Positions ₄ , B ₂ and Dj take place, so that after the expiry of the exposure time required to remove the protective groups, a chain extension can only take place at these locations on the substrate with the provision of a monomeric unit to be guided through the flow chamber.

By means of the procedure outlined here, the light-emitting diode arrangement 1 is used as an array of individual light sources without an individual mask set being required for each substrate carrier or each chip surface 19. Using the computer-assisted individual control of individual light-emitting diodes with respect to the exposure time of the exposure, preselection of the exposure locations, depending on the sequence file stored in the computer 22, small series can also advantageously be synthesized.

The light-emitting diode arrangement 1 controlled by means of the computing unit 22 assumes both the function of the exposure and that of masking the area to be exposed, as a result of which the need to reposition masks can be completely eliminated. Inaccuracies in mask repositioning during the synthesis process using the mask process have in the past led to considerable quality deficiencies in biopolymer components synthesized in this way.

2 shows a synthesized oligonucleotide array, synthesized using an array of 4 × 4 individually controllable light-emitting diodes 2. In addition to the configuration of a light-emitting diode array 1 shown here, this can also have any number, for example 25, 400 or up to several thousand individual ones Contain radiation sources in the form of light-emitting diodes, it being possible to decide whether they can be arranged square, rectangular, ring-shaped or also circular. That in Fig. 2 the array shown is a fluorescence image obtained by hybridization with a fluorescence masked complementary strand probe.

FIG. 3 shows an overview of four surface fluorescence images, each with four different sensitivity levels. A 3 x 3 light-emitting diode array 1 was used to determine a sufficient exposure time. The four images show the same array, recorded in four different sensitivity levels of the detecting scanner.

While there are no signals in the surface fluorescence images 14, 3.1 and 3.2 recorded with low sensitivities 15, 16, these are clearly recognizable in the surface fluorescence images shown in FIGS. 3.3 and 3.4 with higher sensitivity levels 17 and 18, respectively. The intensity of the signals is proportional to the efficiency of the cleavage of the unstable photo protection groups at the respective position on the substrate carrier 12 with a predeterminable irradiation time. The irradiation was carried out for 1, 3, 5, 7, 10, 13, 15, 20 and 30 minutes. The successful removal of the photo protection group due to the irradiation by a covalent attachment of a Cy 5 phosphoramidite became visible after the irradiation.

FIGS. 4.1 and 4.2 show the structure of a sequence on a surface 19 with a light-emitting diode arrangement 1 containing 4 individual light-emitting diodes 2.

On the surface fluorescence images 14, which were recorded with different sensitivity levels 16, 18, the sequence d (CGCTGGAC) was built up at the four positions 19, which are shown brighter, by means of light-controlled DNA chip synthesis. This was a four Individual light-emitting diodes 2 containing light-emitting diode array 1 are used, which emit radiation in the ultraviolet wavelength range. The images shown in Fig. 4.1 and 4.2 were recorded with different sensitivity levels of the scanner. There was a DNA chip synthesis with 2 x 2 UV light-emitting diode arrangement 1 of the sequence CGCTGGAC, which was hybridized with fluorescence-labeled GTCCAGCG with an exposure time of 10 minutes.

Figures 4.1 and 4.2 shown were obtained after hybridization with the complementary 5'-Cy-5 labeled probe after scanning in a fluorescence imaging unit.

LIST OF REFERENCE NUMBERS

1. LED array

2. Single LED

3. Field boundary

4. Control line

5. Resistance

6. Memory cell

7. Memory cell

8. Supply voltage part

9. Grounding

10. PC parallel interface

11. Interface line 1 to 25

12. Slides

13. Side edge

14. Surface fluorescence image

15. Sensitivity level low

16. Sensitivity level higher

17. Sensitivity level high 18. Sensitivity level very high

19. Chip surface A _t LED position

20. Sequence d (CGCTGGAC) B ₃ LED position

21. Sequence position D ₄ LED position

22. Computing unit

Claims

claims

1. A method for the light-controlled synthesis of biopolymers on surfaces, characterized in that selection and activation of areas on a solid support are brought about by imaging an arrangement (1) of electrically controllable light-emitting diodes (2).

2. The method according to claim 1, characterized in that biopolymers are immobilized on slides (12, 19).

3. The method according to claim 1, characterized in that the light-emitting diodes (2) are controlled individually in accordance with a file of sequences (20) stored in a computer (22).

4. The method according to claim 1, characterized in that the individual light-emitting diodes (2) emit high-energy radiation in the UV range.

5. The method according to claim 1, characterized in that the exposure of several areas by the light emitting diodes (2) takes place simultaneously.

6. The method according to claim 1, characterized in that the exposure is carried out sequentially.

7. The method according to claim 1, characterized in that the control of the LEDs (2) of the LED array (1) through the parallel interface (10, 11) of a computing unit (22).

8. The method according to claim 1, characterized in that the substrate for the biopolymers to be synthesized thereon is located in a feed device under a translucent area.

9. The method according to claim 8, characterized in that the chemicals required for the synthesis are offered sequentially and the exposure takes place in the feed device.

10. The method according to claim 1, characterized in that the exposure takes place spatially separated from the chemical synthesis.

11. The method according to claim 2, characterized in that the sequential sequence of the immobilization in the computer (22) is stored in a file.

12. The method according to claim 1, characterized in that suitable optical imaging methods are used for the geometric scaling of the light-emitting diode array (1) on the biopolymer array.

13. Device for light-controlled biopolymer synthesis on slides (12, 19) with an exposure source, characterized in that an exposure arrangement (1) is assigned to a slide (12, 19), which consists of electrically controllable light-emitting diodes (2).