WO2002094454A1

WO2002094454A1 - Fabrication of microdevices for parallel analysis of biomolecules

Info

Publication number: WO2002094454A1
Application number: PCT/US2002/016326
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 micordevices for parallel analysis of biomolecules 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 PARALLEL ANALYSIS OF

BIOMOLECULES

BACKGROUND OF THE INVENTION

Fabrication of microdevices for parallel analysis of biomolecules comprises

providing at least parts of the microfluidic device 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 (CVD) and the

use of homogenously distributed functional groups for defined surface design.

Those microdevices have potential use for parallel 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.

The number of clinical compounds that are available for clinical studies has

dramatically increased over the last years due to successes in combinatorial chemistry. In

parallel, a vast number of potential target molecules were identified due to recent

developments in genomics.

Both, the availability of large drug libraries and the increased number of new target molecules have created a demand for novel technologies for screening of biologically

relevant molecules. For parallel screening of a quantity of biomolecules, a large number of target molecules is generally immobilized to a substrate and a solution containing biomolecules is contacted with the surface. Current techniques often rely on microwell

plates with sample volume in the microliter range. This requires high sample volumes,

high costs and time-consuming processes.

Microdevices for in vitro screening potentially require smaller sample volume and

may provide access to more efficient studies of biomolecules. The field of micro fluidics has witnessed astonishing advances over the last few years such as the development of

micro total analysis systems (μTAS), microfabricated cell sorters, or microseparators for

DNA and proteins (Cremers et al., Analytical Chemistry, 2002). Recently, microfluidic devices have been used for continuous-flow cell-based assays. These devices utilize

electrokinetic pumping and hydrodynamic pressure to manipulate nanoliter-scale fluid

flow with potential advantages such as the reduction in sample consumption, parallel

processing for high-throughput screening, and improved data quality. However, current

microdevices lack the availability of surfaces that are compatible with screening methods

in proteomics. This is even more critical as microdevices are characterized by high

surface-to-volume ratios when compared to conventional systems. In addition,

miniaturized chips were developed for screening of DNA-containing material (e.g. US 5412087, US 5445934 and US 5744305) and have found wide application in DNA

hybridization assays. Those systems developed for DNA cannot immediately be

transferred to protein screening because the used materials, such as silicon or glass, are not

compatible with proteins. Furthermore, the immobilization methods developed for DNA

are not immediately applicable for proteins. In spite of the pronounced emphasis that 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 limit the 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 their favorable mechanical properties and their 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 makes the immobilization of proteins, enzymes or antibodies difficult. Furthermore, polymers maybe hydrophobic and may support non-specific

protein adhesion. They also suffer from a 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 will 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.

The deposition of thin polymer films that establish chemically defined interfaces

offers a unique way to overcome these limitations. Therefore, WO 00/04390 proposes a method for surface modification of polymer-based microdevices comprising of a

nanometer-thin interface that allows immobilization of biomolecules via spacer systems.

However, a surface modification of the bulk material prior to immobilization is still

required. Alternatively, the use of self-assembled monolayers on gold or silicon is

proposed as well. Although well-established, this technique requires previous deposition of gold and is therefore also characterized by the above-mentioned disadvantages. 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 ashing 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 fabrication or incorporation of potentially

harmful chemicals.

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

Surface coating methods include carbon-like diamond coatings (CLD), carbon nitride coating, deposition of several metal layers or simple spin, dip, or spray coating of

polymers. CVD polymerization coatings of paracyclophane or chlorine derivatives

thereof, applied in order to achieve inert surfaces (Swarc, Gorham, Union Carbide) have

excellent homogeneity, adhesion and stability. Recently CVD 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

immobilization of biologically relevant molecules. 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.

PCT/US99/15968 discloses arrays of protein-capture agents, which are useful for

the simultaneous detection of a plurality of proteins. The arrays comprise a thin organic layer that is between 10 and 20 nm thick. The use of monomolecular dimensioned

interlayer is associated with disadvantages described for self-assembled monolayers.

SAM's are restricted to a few substrate materials; porous structures such as foams,

scaffolds or membranes are difficult to process and applications in chemically aggressive

environments such as in vivo are not possible. The herein disclosed methods allow for overcoming these drawbacks.

U.S. Patent No. 6,103,479 (Taylor) discloses miniaturized cell array methods and apparatus for cell-based screening. These devices can be used with methods of performing

high-throughput screening of the physiological response of cells to biologically active

compounds and methods of combining high-throughput with high-content spatial

information at the cellular and sub-cellular level as well as physiological, biochemical and

molecular activities.

Other prior art references, which generally describe cell arrays and methods and

apparatus to use the same include WO 01/07891 (Kapur et al.), WO 00/60356 (Kapur et

al.), U.S. Pat No. 5,776,748 (Singhvi et al.), and WO 00/53625 (Rossi et al.). However, all of these references include multi-step processes and include the use of solvent.

U.S. Patent No. 6,192,168 (Feldstein et al.) describes a reflectively coated optical

waveguide and fluidics cell integration, which includes a waveguide having a patterned,

reflective coating.

Additional references in the field of cell assays include PCT Pub. Nos. WO

00/60356 and WO 01/07891. These disclose methods for making a substrate for selective cell patterning and the substrates themselves; and for array for cell screening and methods of making them, respectively. Arrays of protein-captive agents are useful for simultaneous detection of a plurality

of proteins which are expression products or fragments thereof, of a cell or population of

cells as described in PCT WO 00/04389. The arrays are useful for various proteomic applications including assessing patterns of protein expression and modification in cells.

Biomolecules in the sense of the invention include, but are not limited to peptides, amino acids, proteins, DNA, RNA, nucleotides, adenosine, thymidine, guanosine, cytidine,

uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine, nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl

adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine,

C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,

8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,

biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars

(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified

phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), growth factors, cell adhesion molecules including tenascins, astrotactin, cintactin, NrCAM,

neurofascins, U, Neuroglian, TAG-1, anxonin-1, fascilins, cadherins, selectins, ephirins,

netrins, semaphorines, chemokines, interleucines, neurotrophines, neurotransmitters,

catecholamin receptors, growth factor receptors, amino acid receptors or derivatives

thereof, cytokine receptors, extracellular matrix molecule receptors, integrins, integrin

receptors, cell response modifiers such as chemotactic factors, hormones, hormone receptors, antibodies, lectines, cytokines, leptines, serpines, enzymes, proteases, kinases, sulfotransferases, metalloproteases, phosphatases, hydrolases, transcription factors, DNA

binding proteins or peptides, RNA binding proteins or peptides, cell surface antigens, vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents, plasmids, virologe

proteins such as HFV-protease or hepatitis C virus protease, biological ligands, receptors,

polysaccharides, lipids, antibodies, haptenes, nucleoproteins, glycoproteins, lipoproteins, steroids, or phages whether naturally-occurring or artificially created (e.g., by synthetic or

recombinant methods).

SUMMARY OF THE INVENTION

A one-step CVD 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 cell arrays, immobilization of drugs for tissue engineering, micro-

reactors, surfaces for protein or DNA screening, or electro-optical devices.

When chemically addressable surfaces are needed for the fabrication of

microdevices for parallel analysis of biomolecules, CVD 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 microstructuring 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 CVD process resulting in functionalized coating for parallel analysis of biomoecules. It is another object of this invention to provide the functionalized coating on

essentially any shaped three dimensional or porous structure for parallel analysis of

biomoecules.

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

producing a chemically addressable surface for parallel analysis of biomoecules. 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 of reacting with

functional groups of a target molecule resulting in stable 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 CVD

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

thereof. The fabrication of microdevices for parallel analysis of biomolecules preferably comprises a functionalized polymer that 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, while other repetition units can be variably designed, although para-

xylylene is the mainly suitable repetition unit.

(1)

(2)

(R= 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. Due

to the chemically stable background of the deposited polymer aging effects as a

consequence of interactions with analyte solutions can be reduced or ruled out.

The functional groups can be used for immobilization of capturing biomolecules

for development of bioassays. 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, 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, carrier

protein/substrate, lectine/carbohydrate, protein/carbohydrate, carbohydrate/carbohydrate, cell adhesion molecule/cell surface receptor, 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 described microdevices are especially useful for screening or high-throughput screening 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, trasncription factors, DNA binding proteins or peptides, RNA binding proteins or peptides, cell surface antigenes, virologe proteins such as HlV-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, neurotransmitters, 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/

(ET) 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. Examples for spacers include, but are not restricted to diisocyantes, dicarbxylic

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 in parallel. 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 then becomes

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 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, 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.

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 PDMS device is used for DNA screening including a 64x64 channel PDMS setup with a

depth of ca. 100 μm. Each membrane is built up with 4 different functional groups. 256

membranes are aligned in batch process resulting in 4 different possibilities for DNA screening. The substrates are size: 8x8x3 cm and are made of a material (polydimethylsiloxane, PDMS) that is about 50 times cheaper than silicon. PDMS devices are CVD-coated with poly(amino-p-xylylene) and are allowed to react with a solution of

hexamethylene diisocyanate. Subsequently, they are incubated for 4h in a solution of 2-

aminoethoxyethanol (10 mM in DMF). After rinse several times with PBS patterned

samples are incubated in sterile petri dishes with PBS buffer (pH 7.4) containing 0.1% (w/v) bovine albumin and 0.02% (v/v) Tween 20 for 30 min, and with a solution of

streptavidin (10 mM, Pierce, USA) in PBS buffer containing 0.1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20 for another 60 min. The surface is rinsed several times with PBS and then exposed for 120 min to a solution of Biotin-DNA oligomers (0.5mM)

in TE buffer (lOmM Tris HC1, ImM EDTA, PH = 8). The samples are rinsed in TE buffer.

A drop (10μl) of fluorescence-DNA oligomer solution mixed with lOμl of TE buffer is

dropped onto the surface for 2h. The samples are rinsed very well with TE buffer and

examined by fluorescence microscopy using a HFX-DX fluorescence microscope (Nikon,

Japan).

Application 2

Microfabrication techniques and scale-up by replication have fueled spectacular

advances in the electronics industry, and they are now creating new opportunities for reaction engineering. The term "microreactor" typically relates to a small tubular reactor

for testing catalyst performance.

Microfluidics, the manipulation of liquids and gases in channels having cross-

sectional dimensions on the order of 10-100 μm, will be a central technology in a number

of miniaturized systems that are being developed for chemical, biological, and medical applications. These applications can be categorized into four broad areas: miniaturized analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems

for fundamental research. In order for these systems to be successful, they must have the attributes that are required for the particular application, e.g. optical properties and surface

chemistry, and they must also be fabricated in materials that are inexpensive and rugged and use processes that are amenable to manufacturing. A suitable microreactor design

includes an poly(isocyanato-p-xylylene) coated PDMS substrate. The pieces are aligned and glued together resulting in a simple technique for manufacturing complex devices. A

solution of a biotin-conjugated mouse anti-human monoclonal antibody is flown into the microchannel for 2h. Surfaces exposing patterns are incubated with a suspension of bovine

aortic endothelial cells (BAECs) in serum-free Dulbecco's modified eagle medium

(DMEM). After 24 h, cell attachment is studied by means of immunofluorescence

microscopy. Cell nuclei are stained with bis-benzimide.

Application 3

An electrophoresis chambers designed as a PDMS based device. It is CVD-coated

providing functionalities as of demand. In this case a poly(isocyanto-p-xylylene) was

utilized. Functional groups are used to enhance surface properties (optional). The surface is stable and there is no re-orientation of surfaces due to environment.

Application 4

A silicon micro-reactor is utilized. It includes microscopic features, such as posts

or scaffolds, to increase the surface area. A CVD coating is applied inside the channel, antibodies, ligands etc are 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 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 depositions 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 5

First, a substrate is coated, e.g. silicon wafer with poly(isocyanato-p-xylylene).

Parts of the surface are coated with amino-biotin. The remaining parts are coated with

amino-PEG (optional). Amino or alcohol terminated nanocrystals are attached defining a certain color, e.g. red. The steps are repeated using a different pattern and colors e.g. blue,

yellow. A multi color flat display device with excellent brightness is devised. Waveguides for bioaffinity assays or surface-sensitive optical detection can be

prepared using CVD coating processes as described herein, including all of the benefits of

the new process. The waveguide may include a patterned, reflective coated and an

optically exposed region which is sensitive to analytes.

CVD methods may be used to prepare substrates having channels located therein.

The channeled substrates can then be used in a method where charged entities are moved through the channel under influence of an applied voltage differential.

CVD processes can be used to prepare high-content and high-throughput screening.

The method may include providing a base and preparing a micro-patterned chemical array

using the CVD processes described herein. Unique characteristics of a so-designed device

is the option of various instantly reacting functional groups, a defined and chemically

stable polymer base layer with defined properties, non-degradability, the possibility to

create activity gradients, and the feasibility for patterning.

Drug screening devices can advantageously be prepared using the CVD processes

including coating at least one organic thin film onto all or a portion of a substrate. Then a

plurality of patches can be coated in discrete known regions of the coated substrate

surface. The coated devices can then be used to screen for the specific drugs.

Alternative applications may include decollation of implantable devices, e.g. heart

valves, pacemakers, stents, embolization coils, bone substitution, hip substitution, bone

screws, vascular grafts, etc.; improved scaffold for tissue engineering; plates for in vitro

cell culture; resins for proteins synthesis; resins for protein chemistry; microchip based

diagnostic screenings; protein purification; DNA purification; DNA chips; protein chips; arrays of quantum dots; electro-optical devices; and coating of three-dimensional

structures such as membranes, micro-reactors, micro-channels, foams, scaffold, etc. In addition, it is suitable for three-dimensional substrates; any substrate material,

while SAM's are restricted two a few substrate materials; porous structures such as foams,

scaffolds, membranes; applications in chemically aggressive environments such as in vivo;

drug gradients possible; high-resistance coatings possible; thickness variable from some

20 nm to μm and are easy to scale-up, are inexpensive and have high throughputs.

The substrates may be selected from silicon implants for gene testing, gold coated

silicon for μCP, μ-reactors, polymer surfaces for μCP, coated microfabricated electrodes,

glass slides, paper, metal sheets etc.

Although the present invention has been shown and described with respect to several

preferred embodiments thereof, various changes, omissions and additions to the form and

detail thereof, may be made therein, without departing from the spirit and scope of the

invention. Therefore, it is the object of the claims to cover all such variations and

modifications as come within the true spirit and scope of the invention.

Claims

1. Fabrication of microdevices for parallel analysis of biomolecules comprising a vapor

deposition coating process such that the coating includes functional groups having

sufficient intrinsic reactivity to react with target molecules.

2. Fabrication of microdevices for parallel analysis of biomolecules, 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:

(1)

(2)

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

3. Fabrication of microdevices for parallel analysis of biomolecules, 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 according to claim 7 comprising the use of spacer systems to confine capturing molecules to the surface.

9. Method according to claim 7, wherein at least a part of the capturing molecules specifically binds to biomolecules that are subject to screening.

10. Method according to claim 9, wherein a surface is provided; said surfaces further comprising capturing molecules confined to the surface, which only temporarily bind at least a part of the biomolecules being subject to screening, thereby allowing their subsequent release.

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

12. Method of claim 2, wherein a functionalized polymer is provided; said polymer having a thickness between 20 and 2000 nm.

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

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

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 a patterned surface design with

between 10 and 5000 reactive locations at the surface.

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

microchannels per square centimeter.

30. Article of claim 26 comprising; said article comprising reactive locations that are connected with micorchannels.

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, reaction

microchannel, parallel enrichment channel, 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.