DEVICE AND METHOD FOR THE GENERATION OF MOLECULAR MICROARRAYS

• US 20140038854A1
• Filed: 02/03/2012
• Published: 02/06/2014
• Est. Priority Date: 02/03/2011
• Status: Active Grant

• Current Assignee: Biocopy GmbH
• Original Assignee: Albert Ludwigs University of Freiburg
• Inventors: Roth, Gnter, Burger, Jgen
• Granted Patent: US 9,623,394 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00337 Valves
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0822 : Slides
• CPC Class Codes: B01L 3/508 : rigid containers not provided for above
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites

Abstract

The invention relates to a device and a method for the generation of molecular microarrays. The invention relates therefore to a universal approach for the generation of protein microarrays, DNA microarrays and RNA microarrays (in general nucleic acid microarrays), by production of an output molecule from a template molecule microarray via enzymatic or chemical processes and transfer of the output molecule onto the desired molecular microarray.

Claims

1. Method for producing a molecular microarray, comprising

a) providing a first support surface (template surface), that exhibits one or more template molecules immobilized on its surface, and an opposing second support surface (capture or microarray surface),

b) production of an output molecule from said template molecule via a cell-free enzymatic and/or chemical reaction system,

c) transfer of said output molecule to a second support surface via fluid between the first and second support surface, with a correlation between the location of the template molecules on the first support surface and the deposition of the corresponding output molecules on the second support surface,

wherein

d) assembly of the support surfaces is decoupled from initiation of step b), wherein initiation of step b) is prevented by removable restrictive means.

2. The method according to claim 1, wherein

the removable restrictive means are

a spatial separation between the first support surface and the second support surface, preventing direct physical contact of the two surfaces,

a chemical or energetic environment that blocks the cell-free enzymatic and/or chemical reaction system, such as pH value and/or temperature that limits or blocks the activity of said system, or

an internal or external force field, via an electric or magnetic field and/or potential that blocks the cell-free enzymatic reaction system.

3. The method according to claim 2, wherein

the spatial separation between the support surfaces is a microfluidic incubation chamber (microfluidic gap) formed between opposing separated first and second support surfaces.

4. The method according to claim 3, wherein

the microfluidic incubation chamber does not comprise of a membrane positioned between the first and second support surfaces.

5. The method according to claim 1, wherein

removal of the restrictive means occurs by introduction of cell-free enzymatic and/or chemical reaction system into the microfluidic incubation chamber, thereby inducing production of said output molecule and enabling transfer of said output molecule to said second support surface.

6. The method according to claim 1, wherein

the first support surface and second support surface are held in a fixed position, opposing one another, preferably via a mechanical tension or spring system, before initiation of the production of said output molecule via a cell-free enzymatic and/or chemical reaction system according to step b) of claim 1.

7. The method according to claim 1, wherein

the method can be repeated using a single first support surface multiple times for production of multiple microarrays.

8. The method according to claim 1, wherein

the removable restrictive means relate to chemical blocking agents present on the first and/or second support surfaces, that block the cell-free enzymatic and/or chemical reaction system and/or block binding of the output molecule to the second support surface, which can be modified and/or removed as required to initiate the method, or depletion or restriction of essential chemical compounds for the cell-free enzymatic reaction system.

9. The method according to claim 1, wherein

the blocking of the cell-free enzymatic and/or chemical reaction system relates to use of light cleavable chemical substituents attached to essential —

OH groups of reaction components, wherein treatment with light releases the reaction component and enables reaction initiation, or binding and/or capture of essential reaction components to either the first or second surface, such as ATP, essential salts or coenzymes such as vitamins or metal ions, so that reaction initiation occurs only upon filling or an external impulse.

10. The method according to claim 1, wherein

the removable restrictive means relate to molecular switching of molecules from active to inactive state, or from inactive to active state,

by pH-change, inducing a functional change in pH-sensitive molecules, for example through light treatment of a surface coated with titanium dioxide leading to generation of H+ ions and subsequent pH change,

change in static and/or dynamic electrical and/or magnetic fields, inducing a change in charged, dielectric or magnetic molecules or surface properties,

change in temperature, inducing a change of the molecular structure or dynamics, such as reduced temperature leading to inactivity of a DNA or RNA polymerase, wherein upon warming reaction initiation occurs,

illumination, inducing a change of blocked, photo-sensitive or caged molecules by light-induced reactions, such as use of light cleavable chemical substituents attached to essential reaction components, whereby treatment with light releases the reaction component and enables reaction initiation, and/or release of caged compounds, such as light induced release of caged biotin or other molecules,

or a combination thereof.

11. The method according to claim 1, wherein the template molecule is a nucleic acid or nucleic acid-like molecule, such as DNA, RNA, genomic DNA, cloned DNA fragments, plasmid DNA, cDNA or cDNA libraries, PCR products, synthetic DNA or DNA oligonucleotides, mRNA or synthetic RNA.

12. The method according to claim 1, wherein

the cell-free enzymatic reaction system is

a DNA-polymerase or a DNA-amplificating enzyme or enzyme system,

a RNA-polymerase or a RNA-amplificating enzyme or enzyme system,

a reverse transcriptase or a RNA-to-DNA transcribing enzyme or enzyme system,

a protein synthesizing system or a cell free expression mix, such as an enzyme mix required for transcription of DNA to RNA and translation of RNA to protein, such as cell-free lysate selected from a prokaryotic or eukaryotic system, such as E. coli, bacterial origin, rabbit reticulocyte, insect origin, human and wheatgerm.

13. The method according to claim 12, wherein

the cell-free enzymatic reaction system is a DNA-polymerase, the output molecules are DNA and a DNA microarray is generated on the second support surface.

14. The method according to claim 12, wherein

the cell-free enzymatic reaction system is a RNA-polymerase, the output molecules are RNA and a RNA microarray is generated on the second support surface.

15. The method according to claim 12, wherein

the cell-free enzymatic reaction system is a reverse transcriptase, the output molecules are DNA and a DNA microarray is generated on the second support surface.

16. The method according to claim 12, wherein

the cell-free enzymatic reaction system is a protein synthesizing system or a cell free expression mix, such as an enzyme mix required for transcription of DNA to RNA and translation of RNA to protein, the output molecules are protein and a protein microarray is generated on the second support surface.

17. A molecular array produced by the method according to claim 1.

18. Device for the production of a molecular microarray, preferably for carrying out the method according to claim 1, comprising

a) a first support surface (template surface) that exhibits one or more template molecules immobilized on its surface,

b) a second support surface (capture or microarray surface) assembled with said first support surface,

c) wherein a microfluidic incubation chamber (microfluidic gap) is formed between physically separated and opposing first and second support surfaces for a cell-free enzymatic and/or chemical reaction system, thereby decoupling assembly of the support surfaces from initiation of cell-free enzymatic and/or chemical reaction system,

d) a fluid inlet and/or outlet into the incubation chamber,

e) holder for the two opposing support surfaces in a fixed position, and

f) means for maintaining the incubation chamber as a space between the two opposing support surfaces.

19. The device according to claim 18, wherein

the microfluidic incubation chamber does not consist of a membrane positioned between the first and second support surfaces.

20. The device according to claim 18, wherein

the means for maintaining the incubation chamber as a space between the two opposing support surfaces is a spacer between the first and second support surfaces.

21. The device according to claim 18, wherein

the means for maintaining the incubation chamber as a space between the two opposing support surfaces is one or more three-dimensionally structured (3D) flow cells, preferably of synthetic polymers, such as thin film polymer materials or Polydimethylsiloxane (PDMS).

22. The device according to claim 18, wherein

the fluid inlet and/or outlet is suitable for the cell-free enzymatic and/or chemical reaction system to be pumped or pipetted into and/or out of the incubation chamber.

23. The device according to claim 18, wherein

the template molecule is a nucleic acid or nucleic acid-like molecule, such as DNA, RNA, genomic DNA, cloned DNA fragments, plasmid DNA, cDNA or cDNA libraries, PCR products, synthetic DNA or DNA oligonucleotides, mRNA or synthetic RNA.

24. The device according to claim 18, wherein

the spatial separation between the support surfaces is less than 100 micrometer in height, preferably less than 80 micrometer, such as 65 micrometer, preferably less than 60 micrometer, or more preferably less than 40 micrometer, such as 20 micrometer and less.

25. The device according to claim 18, wherein

said first and second support surfaces are glass, plastic, nylon or other type of natural or synthetic polymer or membrane, for example Polydimethylsiloxane (PDMS).

26. The device according to claim 18, wherein

the first and/or second support surface is a standard glass slide(s) suitable for use in microscopy, such as with dimensions of 76×

26×

1 mm³.

27. The device according to claim 18, wherein

the device is of handheld size, preferably of 60-140 mm, 80-120 mm or more preferably of 105 mm in length, and preferably of 30-90 mm, 40-80 mm, or more preferably of 60 mm in width.

28. The device according to claim 18, wherein

the holder for the two opposing support surfaces in a fixed position relate to mounting brackets (holders), positioned either as upper, lower, or side brackets, positioned in relation to the two support surfaces.

29. The device according to claim 18, wherein

either the support surfaces or the mounting brackets (holders) are held in place by mechanical tension, magnetism, a spring system, guiding rails for the surfaces thereby holding the two support surfaces in a fixed position.

30. The device according to claim 18, wherein

said second support surface is pre-coated with an immobilization agent configured to covalently or non-covalently attach the output molecule to the surface.

31. The device according to claim 18, wherein

the immobilization agent is a protein immobilization agent, such as an antibody, configured to covalently or non-covalently attach to the expressed protein, a polyhistidine sequence such as hexahistidine, wherein said protein immobilization agent is a chelating agent such as Ni-NTA, a peptide, domain or protein, wherein said protein immobilization agent is an antibody specific to said tag and/or a biotin-binding molecule, such as avidin.

32. The device according to claim 18, wherein

the first support surface is

a microarray of nucleic acids or nucleic acid-like molecules,

a sequencing chip exhibiting nucleic acids,

a spatially defined distribution of nucleic acids on a surface,

a spatially defined distribution of nucleic acids on a bead array or a structured surface, or

a spatially defined distribution of liquid or solid material containing nucleic acids.

33. System for the production of a molecular microarray, comprising

a) a first support surface (template surface) that exhibits one or more template molecules immobilized on its surface,

b) a second support surface (capture or microarray surface) assembled with said first support surface, whereby a microfluidic incubation chamber (microfluidic gap) is formed between physically separated and opposing first (template) and second (capture or microarray) support surfaces,

c) a fluid inlet and/or outlet into the incubation chamber,

d) holder for the two opposing support surfaces in a fixed position, and

e) means for maintaining the incubation chamber as a space between the two opposing support surfaces,

wherein

f) a microarray is formed by production of an output molecule from the template molecule and transfer of said output molecule to the surface of the second support surface (capture or microarray surface) after introduction of cell-free enzymatic and/or chemical reaction system into the microfluidic incubation chamber, thereby inducing production and transfer of said output molecule to said second support surface.