WO2002094456A1

WO2002094456A1 - Fabrication of microdevices for separation of biomolecules in an electrical field

Info

Publication number: WO2002094456A1
Application number: PCT/US2002/016520
Authority: WO
Inventors: Joerg Lahann
Original assignee: Joerg Lahann
Priority date: 2001-05-22
Filing date: 2002-05-22
Publication date: 2002-11-28

Abstract

Fabrication of microdevices for separation of biomolecules in an electrical field comprising a vapor deposition coating process such that the coating includes polymer interfaces containing chemical groups having sufficient intrinsic reactivity to react with target molecules.

Description

FABRICATION OF MICRODEVICES FOR SEPARATION OF BIOMOLECULES IN AN

ELECTRICAL FIELD

BACKGROUND OF THE INVENTION

The fabrication of microdevices for separation of biomolecules in an electrical field is

disclosed comprising coating of at least parts of the microdevice with a functionalized polymer. The entire functionalized polymer or parts thereof can subsequently be modified

by an immobilized biological coating, which specifically recognizes at least a part of the

biomolecules being subject to detection. The invention further relates to the field of

polymer coating of functional groups made by chemical vapor deposition (CND) and the

use of homogenously distributed functional groups for defined surface design.

Those microdevices have potential use for screening of a quantity of biologically active

molecules and are in immediate context with pharmaceutical technologies in the fields of

drug discovery, proteomics, genomics, high-throughput screening and clinical diagnostics. Electrophoresis is a critical tool in biotechnology for sample preparation, isolation, and

detection. Often used sample material includes nucleotides, DΝA, proteins, peptides, or

polysaccharides. In the field of electrophoresis, capillary electrophoresis (CE) is especially

important, where charged molecules are manipulated within a capillary due to the

influence of an applied electrical field. Even more advantageous is the micro capillary electrophoresis (MCE), where the capillary electrophoresis chamber is replaced by a

microchannel. Both, CE and MCE find increasing applications for analysis, biomedicine, pharmaceutical

technology, biology, environmental analysis, and food technology.

Devises for MCE are known from US 5126022, US 5296114, or US 5180480. The use of

polymers for fabrication of MCE devices is proposed in US 588246. Although a

pronounced emphasis lies on devices made from silicon or glass, these may not be the first choice for many microfluidic applications especially in biology or medicine. Several

properties of silicon and glass could limit their use for microfluidic devices, including: (i) limited biocompatibility, (ii) intrinsic stiffness, (iii) unfavorable geometry, and (iv)

incompatibility with soft materials needed e.g. for incorporation of valves. New materials and novel immobilization strategies are therefore necessary to extend the use of

microsystems towards proteins. The use of polymers for manufacturing microdevices is

proposed in US 588246. Polymers are often considered as an alternative due to its

favorable mechanical properties and its straightforward manufacturing by rapid prototyping. However, polymers are incompatible with organic solvents required for

organic synthesis or analysis. Moreover, the absence of surface functional groups

difficulties the immobilization of proteins, enzymes or antibodies. Furthermore, polymers

are hydrophobic and supports non-specific protein adhesion. They also suffer from the lack

of defined and constant surface properties under ambiguous conditions. Due to the high

surface-to-volume ratios in microfluidic devices, even slight inhomogeneities in the

surface result in malfunction. While surface modification of glass substrates via silane

chemistry has been well established, simple, well-defined surface modification protocols

for polymers are still in their infancies. For this reason, US 5935409 proposes a procedure

for surface modification of electrophoresis chambers. The surface modification is troublesome, poorly reliable and expensive. US 5322608, US 5447616, and EP 452055B1 similarly describe fabrication of electrophoresis chambers using surface modification. EP

665340B1 reports surface modification of a polymer device by incubation with harsh

chemicals. The resulting functional groups are then used for further modification. Other

methods for surface modification of materials are plasma aching and plasma

polymerization (see Yasuda or EP 0519087 Al), laser treatment, or ion beam treatment. The underlying mechanisms are often poorly understood and these methods are

characterized by side reactions including the production or incorporation of potentially harmful chemicals.

Rather than pure surface modification, surface coating is the method of choice for

some applications. Surface coating methods include carbon like diamond coatings (CLD),

carbon nitπde coating, deposition of several metal layers or simple spin, dip, or spray

coating of polymers. CND polymerization coatings of paracyclophane or chlorine

derivatives thereof, applied in order to achieve inert surfaces (Swarc, Gorham, Union

Carbide; US Patent application 20020050456) have excellent homogeneity, adhesion and

stability. Recently CND coating of functionalized paracyclophanes has been used in order

to immobilize bioactive proteins (Lahann Biomaterials 2001, Hocker DE 19604173 Al).

This coating procedure developed to be a one-step coating and functionalization

method offers a wide range of applications since good bulk properties of a material has

been maintained combined with enhanced contact properties. The 'activation' of surfaces

with bivalent spacer molecules offers the opportunity of further modification such as drug

immobilization. By using the interfaces for immobilization of proteins, cell receptors,

cytokines, inhibitors etc., bioactive surfaces that interact with the biological environment in a defined and active matter can be achieved. Electrophoresis is an indispensable tool of biotechnology as described in

PCT Pub. No. WO 99/40174. The devices are used in a variety of applications and

preparation of pure samples of nucleic acids, proteins, carbohydrates, the

identification of a particular analyte in a complex mixture and the like. They are

also used in capillary electrophoresis (CE) and microchannel electrophoresis

(MCE). These methods are used for industrial processes and basic research

including analytical, biomechanical, pharmaceutical, environmental, molecular,

biological, food and clinical applications.

U.S. Patent Nos. 5,858,188 and 5,935,409 further describe microchannels

and their use in electrophoresis and processes for recovering metal values from

metal sources containing more than one metal.

SUMMARY OF THE INVENTION

A one-step CND coating process is disclosed such that the coating has polymer interfaces

that contain chemical groups having sufficient intrinsic reactivity to react with target

molecules. The highly reactive surfaces are useful for several applications such as the

manufacturing of electrophoresis devices, micrototalanalysis systems, immobilization of

drugs for tissue engineering, micro-reactors, or devices for protein or DΝA screening.

When chemically addressable surfaces are needed for the production of microdevices for analysis of biomolecules, CND polymerization of functionalized

[2.2]paracyclophanes can be utilized. The technique has been used for coating several

materials with polymers. Since the coating step is substrate-independent, the technology provides a generic approach to surface engineering of microdevices. While overcoming restrictions associated with gold/alkanethiolates-based techniques, the technology maintains intrinsic advantages of soft lithography, e.g. accuracy, broad availability, and

low costs.

It is an object of this invention to provide a one step CND process resulting in

functionalized coating of microdevices for separation of biomolecules in an electrical

field.

It is another object of this invention to provide the functionalized coating on

essentially any shaped three dimensional or porous structure.

It is another object to provide a simple, inexpensive quick scale-up method of

producing a chemically addressable surface for separation of biomolecules in an electric

field.

Additionally it is another object to provide applications for the herein disclosed

methods.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Universal applicability of the reactive coating to various substrates, such as

polymers, metals, or composites makes the procedure described below attractive for

fabrication of microdevices for parallel analysis of biomolecules.

Generally, the interfaces contain functional groups, that are capable to react with

functional groups of a target molecule resulting in stable or metastable chemical linkages and are produced by chemical vapor deposition. The reaction of the interface with the

drug may or may not take advantage of bivalent linker molecules. The reaction of the

interface with the drug may be carried out in aqueous solution ideal for applications associated with proteins, peptides or DNA. The monomer units may be achieved either by

thermal or photochemical activation of suitable precursors (usually paracyclophanes) in a

CND process. All interfaces are preferably based on poly(para-xylylene)s or copolymers

thereof. The interface are built up by polymers that contain one or more different

repetition units, where at least one of the repetition units is prepared from precursors of the type of structure (1) and/or (2), while other repetition units can be variably designed,

although para-xylylene is the preferred repetition unit. Preferred examples are

summarized as examples 1 to 45.

Whereas X i preferably one of th following Br, I, Ν; acetoxy

(1)

(2)

(whereas R is one of the group consisting of hydrogen, C1-C4 alkyl groups, aryl

groups)

Depending on the used polymer, the required temperatures for monomer creation are

between 400 and 1000 °C and the pressures are below 500 Pa.

The proposed procedure for coating of microdevices with functionalized polymers provides an increased surface concentration of functional groups with a defined and

controlled ratio when compared to conventional methods such as plasma treatments. The

functional groups establish an elecrophoretically active, charge layer and defines the interaction with charged molecules in the solution. It further establishes constant flow

parameter, such as a constant electrosmotic flow. Physico-chemical properties such as

zeta-potential or surface tension are constant and truly defined by the functionalized

polymer coating. Due to the rigid background of the deposited polymer, aging effects as a

consequence of interactions with analyte solutions can be ruled out. Furthermore, the functional groups can be used for immobilization of capturing

biomolecules. Capturing biomolecules shall comprise those molecules that are confined at

a surface and bind at least a part of the biomolecules that are subject to screening.

Capturing biomolecules include biological ligands, receptors, cell adhesion molecules,

antibodies, haptenes, lectines, carbohydrates, DNA, RNA, artificial receptors, etc. A

variety of capturing biomolecules is known to an expert to the field; some of them are disclosed in WO 00/04390. The binding event between capturing biomolecules and

biomolecules allows the isolation of a given sort of biomolecules. Therefore, the capturing biomolecules must posses selectivity towards a specific biomolecule or at least a class of

biomolecules. Suitable binding pairs can be antibody/antigene, antibody/heptene,

enzyme/substrate, integrin extracellular matrix component, biomolecule/cell, cell/cell, cell

adhesion molecule/ cell surface receptor, carrier protein/substrate, lectine/carbohydrate,

protein/carbohydrate, carbohydrate/carbohydrate, receptor/hormone, receptor/cytokine, protein/DNA, protein/RNA, peptide/DNA, peptide/RNA, two DNA single strains,

DNA/RNA, DNA/ DNA, where either of both partners of these couples may serve as capturing biomolecule.

The describe microdevices are especially useful for characterization of biomolecules among a biological class of biomolecules. Those biological classes include growth factors, neurotransmitters, catecholamin receptors, growth factor receptors, amino acid receptors or derivatives thereof, cytokine receptors, extracellular matrix molecule receptors, integrins, integrin receptors, hormones, hormone receptors, antibodies, lectines, cytokines, leptines, serpines, enzymes, proteases, kinases, phosphatases, hydrolases, transcription factors, DNA binding proteins or peptides, RNA binding proteins or peptides, cell surface antigenes, virologe proteins such as HIV-protease or hepatitis C virus protease, or phages. Classes of drugs that can be used in the practice of the present invention include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non- steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, and imaging agents. A more complete listing of classes and specific drugs suitable for use in the present invention may be found in "Pharmaceutical Substances : Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", Edited by Susan Budavari et al, CRC Press, 1996, both of which are incorporated herein by reference.

In another embodiment of the invention, the polymer coating may be used to bind

artificial or natural molecules that change the surface properties of the microdevice or

increase the surface area exposed to the analyte solution. Useful molecules include

hydrophobic molecules as well as hydrophilic molecules, such as hydrogels. Especially,

temperature-sensitive materials, such as poly(N-isopropylacrylamide), ethylene oxide/

propylene oxide co-polymers (e.g. Pluronics ), or temperature sensitive proteins or

peptides - natural or synthetic - can be bound to the microdevice via functionalized

polymer coating. Natural hydrogels may include Collagen, Laminin-containing hydrogels,

Fibrin, Elastin, Agerose, Aga, or mixtures thereof. In another embodiment of the invention, spacer systems may be used to bind

molecules. Spacers include but are not restricted to diisocyantes, dicarboxylic acid

chlorides, dioles, diamines, dithiols oder dicarboxylic acids and their active esters. A

spacer is any molecule that allows for chemical connection between surface and target

molecule. The binding occurs via chemical interactions, such as covalent bonding.

Due to the mild character of the deposition process, side reactions are suppressed and the deposited films are homogeneous with respect to their chemical structure and topology - unless otherwise intended. Gradients may be achieved by establishment of

temperature gradients at the substrate being subject to coating. Another advantage of the

disclosed method is that straightforward synthesis and selection of appropriate precursor

allows the establishment of different functional groups beside each other. This feature is

especially crucial, when immobilization of more than one type of biomolecules to the same

substrate is intended. Spatially directed immobilization of biomolecules becomes than

possible. Furthermore, the disclosed invention provides a defined chemical surface even to those microdevices that are composites of different starting materials.

When depositing polymers from precursors based on the general structures (1) or

(2), we found temperatures between 400 und 900 °C and pressures below 150 Pa suitable for activation of the precursor, while deposition was best conducted at temperatures below

160 °C. The there like deposited polymers allow binding of biomolecules - direct or via

spacers. The functionalized coating can be used for microdevices made of different

materials, such as polymers, composites, silicon, semiconductors, glass, or metal.

Furthermore, the once deposited film may be subject to further modification using

conventional methods such as plasma etching with cold plasmas, such as oxygen, water, ammoniac, argon, sulfur dioxide, nitrogen, hydrogen plasmas or mixtures thereof. In another embodiment of the invention, functionalized polymer coatings may be

prepared by co-polymerization of precursors of the general structures (1) or (2) with

precursors of the general structures (3) and/or (4). Those co-polymers were found to be

suitable functionalized polymers for fabrication of microdevices for parallel analysis of biomolecules. They further allow specific tailoring of physical and or chemical surface

properties including topology as required by a given application.

(3) (4)

wherein R_n (n=l , 2,3,4) may be equal or different and may be selected from the group consisting of hydrogen, C1-C4 alkyl, aryl, a ine, alcohol, ether, ethylene glycol, cyclic ether, thioether, crown ether, primary amide, secondary amide, ethylene glycol containing primary amide, ethylene glycol containing secondary amide, urethane, nitrile, isonitrile, nitrosamine, lactone, ethylene glycol containing urethane, carbamate, ethylene glycol containing carbamate, lactam, imine, hydrazone, ester, ethylene glycole containing ester, nitro compounds, nitrile, halo, organic radical, metalized group, acid halide group, isocyantate, thioisocyante, groups of the general nature CO(O-M-A) (with M: C1-C4 aliphatic or aromatic group and A: e.g. hydrogen, hydroxyl-, amino-, or carboxy groups), sulfur-containing groups (e.g. sulfonic acid, thioether, sulfonate, or sulfate ester group), silicon-containing group (e.g. silyl or silyloxy), or sugar derivatives. Prior to coating in the vapor deposition process, pretreatment of the substrate may

be used to improve adhesion behavior. The method of choice is mainly depending on the

type of substrate and all methods well-known to a person skilled in the field of adhesion

improvement may be applied. Especially a pretreatment with cold gas plasmas including, but not limiting to oxygen, hydrogen, nitrogen, ammoniac, carbon dioxide, ethylene,

acetylene, propylenes, butylenes, ethanol, acetone, sulfur dioxide plasmas; or mixtures thereof have proven to be advantageous in improving the adhesion behavior of the

deposited polymer coatings.

Application 1

A silicon micro-reactor is utilized. It include hosts, scaffolds etc. to increase

surface area. A CND coating is applied inside the channel. Antibodies, ligands etc are

selectively attached to each channel. Screening of solution of labeled proteins is

conducted by flowing the solutions in individual channel and then there is a read out. It is

possible to create complex geometries possible, applications for diagnostics, proteomics, and drug screening. The fabrication of the microchannels is based on the techniques of

soft lithography. The master used to cast the PDMS microchannel is fabricated using

photolithographic technique. Photolithography seems to be the most convenient method

for generating patterned microchannels. Once a master is fabricated, the channels are

formed in PDMS by replica molding. Replica molding is simply the casting of prepolymer

against a master and generating a negative replica of the master in PDMS. The PDMS is cured in an oven at 60°C for 6 h at least, and the replica is then peeled from the master.

Patterned deposition of materials in small (<100μm) features is important for applications

of miniaturized systems in biochemistry and cell biology. Patterned attachment of cells is also important in cell-based sensors where the reaction of cells to stimuli in specific areas

of a device is necessary for detection of species of interest.

A solution of (+)biotinyl-3,6,9-trioxaundecanediamine (80% ethanol / 20% DMF)

is used to flow for 4h through the microchannels which are coated with (1) reactive

coating and (2) After flushing for 3. min with PBS containing 0.1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20, a fluorescein-conjugate streptavidin solution is guided through the channel for lh.

Application 2

A silicon micro-reactor is utilized. It includes hosts, scaffolds etc. to increase

selectively attached to each channel. Screening solution with tagged proteins flows in

each channel and then there is a read out. It is possible to create complex geometries

possible, applications for diagnostics, proteomics, drug screening. The fabrication of the

microchannels is based on the techniques of soft lithography. The master used to cast the

PDMS microchannel is fabricated using photolithographic technique. Photolithography seems to be the most convenient method for generating patterned microchannels. Once a

master is fabricated, the channels are formed in PDMS by replica molding. Replica

molding is simply the casting of prepolymer against a master and generating a negative

replica of the master in PDMS. The PDMS is cured in an oven at 60°C for 6 h at least, and

the replica is then peeled from the master. Patterned deposition of materials in small

(<100μm) features is important for applications of miniaturized systems in biochemistry

and cell biology. Patterned attachment of cells is also important in cell-based sensors where the reaction of cells to stimuli in specific areas of a device is necessary for detection

of species of interest.

A solution of (+)biotinyl-3,6,9-trioxaundecanediamine (80% ethanol / 20% DMF)

coating and (2) After flushing for 3. min with PBS containing 0.1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20, a fluorescein-conjugate streptavidin solution is guided

through the channel for lh.

Claims

1. Fabrication of microdevices for separation of biomolecules in an electrical field comprising a vapor deposition coating process such that the coating includes polymer

interfaces containing chemical groups having sufficient intrinsic reactivity to react with

target molecules.

2. Fabrication of microdevices for separation of biomolecules in an electrical field,

wherein a functionalized polymer is prepared from precursors of the general structures (1) and/or (2) by activation at temperatures between 450 and 1000 °C and reduced pressures

below 500 Pa and deposition at lower temperatures on at least parts of the microdevice, with:

Whereas X is one of the following Br, I, N₃, acetoxy

(1)

(2)

(R= hydrogen, C1-C4 alkyl groups, aryl groups)

3. Fabrication of microdevices for separation of biomolecules in an electrical field,

wherein the coatings are based on poly[para-xylylenes]s or copolymers thereof.

4. Method of claim 2, wherein [2.2]paracyclophanes are polymerized during the chemical

vapor deposition process.

5. Method of claim 2, comprising activation of precursors of the general structures (1) or

(2) at temperatures between 600 and 900 °C and pressures below 100 Pa and deposition

of the polymers at temperatures below 160 °C.

6. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

used for confinement of capturing molecules.

7. Method of claim 2, wherein a functionalized polymer is provided; said polymer reacting

with functional groups of target molecules resulting in stable linkages.

8. Method of claim 7 comprising the use of spacer systems to confine capturing molecules

to the surface.

9. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

transparent.

10. Method of claim 2, wherein a functionalized polymer is provided; said polymer having

a thickness between 20 and 2000 nm.

1 1. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

used for confinement of molecules that change surface tension of the luminal surface.

12. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

used for confinement of molecules that change the area of surface interacting with the

analyte solution.

13. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

used for confinement of hydrogels.

14. Method of claim 2, wherein a functionalized polymer is provided; said polymer used

for confinement of polyelectrolytes.

15. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

used for confinement of temperature-sensitive molecules.

16. Method of claim 7 comprising at least a part of the capturing molecules to specifically

bind to biomolecules that are subject to screening.

17. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

further modified by plasma treatment.

18. Method of claim 2, wherein a functionalized polymer is provided; said polymer being further modified by treatment with chemicals.

19. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

further modified by treatment with a high energy source.

20. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

precursors of the general structures (3) and/or (4).

(3) (4)

wherein R_n (n=l , 2,3,4) may be equal or different and may be selected from the group consisting of hydrogen, C1-C4 alkyl, aryl, amine, alcohol, ether, ethylene glycol, cyclic ether, thioether, crown ether, primary amide, secondary amide, ethylene glycol containing primary amide, ethylene glycol containing secondary amide, urethane, nitrile, isonitrile, nitrosamine, lactone, ethylene glycol containing urethane, carbamate, ethylene glycol containing carbamate, lactam, imine, hydrazone, ester, ethylene glycole containing ester, nitro compounds, nitrile, halo, organic radical, metalized group, acid halide group, isocyantate, thioisocyante, groups of the general nature CO(O-M-A) (with M: C1-C4 aliphatic or aromatic group and A: e.g. hydrogen, hydroxyl-, amino-, or carboxy groups), sulfur-containing groups (e.g. sulfonic acid, thioether, sulfonate, or sulfate ester group), silicon-containing group (e.g. silyl or silyloxy), or sugar derivatives.

21. The method of claim 2 comprising a pre-treatment of the surface with a gas plasma

prior to deposition of the functionalized coating.

22. The method of claim 2 comprising coating of a microdevice made from polymers.

23. The method of claim 2 comprising coating of a microdevice made from

polydimethylsiloxane.

24. The method of claim 2 comprising coating of a microdevice made from a

poly(acrylate) and/or poly(methacrylate).

25. The method of claim 2 comprising coating of a microdevice made from glass, silicon

and/or silicon dioxide.

26. Microdevice for separation of biomolecules in an electrical field, said microdevice comprising a polymeric coating for capturing molecules at the surface, which bind at

least a part of the biomolecules being subject to screening only temporarily allowing

their subsequent release.

27. Article of claim 26 comprising a modular design of the microdevice.

28. Article of claim 26 comprising; said article comprising at least one electrophoresis

chamber.

29. Article of claim 26 comprising; said article comprising at least on capillary.

30. Article of claim 26 comprising; said article comprising at least one microchannel.

31. Article of claim 26 comprising; said article comprising between 2 und 1000

microchannels per square centimeter.

32. Article of claim 26 comprising; said article comprising at least one of the following

elements in variable quantity and geometry: inlet reservoir, outlet reservoir,

electrophoresis chamber, parallel enrichment channel, or electrodes.

33. Method of claim 2, wherein a functionalized polymer is provided; said polymer covering only a part of the microdevice surface.

34. Method of claim 2 comprising more than one functionalized polymer coating deposited at different regions of the device surface.

35. Method of claim 2, wherein a functionalized polymer is provided; said polymer being

deposited in different microchannels.

36. Method of claim 2, wherein a functionalized polymer is provided; said polymer comprising an anisotropic distribution of chemical groups on the surface.

37. Method of claim 36, wherein a functionalized polymer is provided; said polymer comprising a chemical and/or biological gradient.