Microfluidic device comprising a multitude of micropillars

FIELD OF THE INVENTION

The present invention is directed to the field of microfluidic devices, especially to the field of microfluidic devices for handling biomolecules in aqueous solution

BACKGROUND OF THE INVENTION

The present invention is directed to microfluidic devices. In the field of microfluidic devices, many approaches have been investigated to build devices which comprise a plurality of "micropillars". E.G. in B. He et al, Analytical Chemistry, Vol. 70, Nr.18 (1998), 3795, the fabrication of collocated monolith support structures is described. Many of these approaches involve anorganic material, e.g. the approach from Mogensen et al. as presented on the Microscale Bio Separation Conference 2007 in Vancouver.

However, there is still a need for alternative microdevices, where porous elements are closely packed and integrated into a microfluidic device as well as easy and cost effective method for making these devices.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a device which is at least partly able to fulfill said needs and which especially can be made in an efficient and easy manner.

This object is solved by a substrate material according to claim 1 of the present invention. Accordingly, a microfluidic device comprising at least one substrate and a multitude of porous elements substantially made out of an polymeric organic material and attached to said substrate, whereby at least >95 % of the outer shell surface of at least >95 % of all porous elements is covered with open pores and has an open outer shell surface porosity of at least >20 % and <90%.

The term "porous elements" especially includes elements such as

micro -pillars and micro-beads which may be of any suitable form.

It should be noted that term "attached" especially includes that there are several further layers or materials between the substrate and the porous elements. Actually this is one preferred embodiment of the present invention. However, the porous elements may be provided on said substrate "directly".

The term "outer shell surface" in the sense of the present invention is defined as the outer surface of the porous element(s) which is not attached to said substrate and has a typical layer thickness of a few hundred nanometer.

In the context of the present invention, the area within the porous elements from a depth of 1 μm on is to be referred as "bulk".

The term "open outer shell surface porosity of X%" especially means and/or includes that X% of the outer shell surface layer is covered with pores. Alternatively, the term "open outer shell surface porosity of X%" also especially means and/or includes that X% of the volume of the outer shell surface layer is empty.

The term "open outer shell surface porosity" especially includes pores in the outer shell surface are connected to the pores of the bulk of said porous elements.

Such a device has shown for a wide range of applications within the present invention to have at least one of the following advantages:

Due to the open porosity of the porous elements, the suitability of the device, especially for chromatography or filtering purposes can be greatly enhanced.

It has been shown that such a device may be built in an easy and efficient way (as will be shown later on).

According to an embodiment of the present invention, at least >95 % of the outer shell surface of at least >95 % of all porous elements is covered with open pores and has an open outer shell surface porosity of at least >30 % and <80%, more preferred >40 % and <70%. According to an embodiment of the present invention, the average pore size of the pores of said porous elements is ≥lOnm and <5μm, preferably

between 50nm and 2μm, most preferably between lOOnm and lμm.

According to an embodiment of the present invention, at least >95 % of the outer shell surface of at least >95 % of all porous elements has an open outer surface porosity, which differs from the open porosity of the bulk material of said porous element by < than a factor of 3, preferably < than factor of 2, most preferably < than a factor of 1.5.

It has been shown in practice that by doing so, for a wide range of applications within the present invention, the purposes (e.g. as described above) of the substrate material may be greatly enhanced. According to an embodiment of the present invention, a substantial part, preferably >30%, more preferred >60% of the bulk of said porous elements has an open porosity of >20% and <90%, according to an embodiment of the present invention, a substantial part preferably >30%, more preferred >60% of the bulk of said porous elements has an open porosity of >30% and <80%, according to an embodiment of the present invention, a substantial part of the bulk of said porous elements has an open porosity of >40% and <70%.

In the sense of the present invention, the term "open porosity" in the context of the "bulk" especially means or includes the fraction of the total volume in which fluid flow is effectively taking place. The pores can according to an embodiment of the present invention be arranged in a periodic pattern or according to an embodiment of the present invention just randomly distributed.

According to an embodiment of the present invention, the average pore size of the inner pores of said porous elements is essentially the same as the average pore size of the outer pores of said porous elements.

According to an embodiment of the present invention, the average pore size distribution within the porous elements (FWHM) is < 2 * the average pore size.

According to an embodiment of the present invention, the average specific surface area of the porous elements is >30 m ² /g. By doing so, the feasibility

of said porous elements can for many applications greatly be enhanced. The average specific surface area is measured by BET adsorption measurements.

It should be noted that in the context of the present invention, the "average specific surface area" may be also called "inner surface area" and especially describes the accessible inner surface (e.g. for a fluid) of said porous elements.

Preferably the average specific surface area of the porous elements is >35 mVg, more preferred >40 m ² /g and most preferred >45 m ² /g.

According to an embodiment of the present invention, the average open porosity of the multitude of porous elements is >50%. In this context, the term "open porosity" especially means or includes the fraction of the total volume in which fluid flow is effectively taking place.

In the sense of the present invention, the term "porosity" especially means or includes the ratio of the volume of all the pores or voids in a material to the volume of the whole. In other words, porosity is the proportion of the non-solid volume to the total volume of material. In the sense of the present invention porosity is especially a fraction between 0% and 100%, e.g. typically ranging from less than 1% for solid granite to more than 90% for some materials like cotton.

According to an embodiment of the present invention, the average open porosity of the multitude of porous elements is > 60%, preferably > 70%, more preferably > 80% and most preferred > 90%.

According to an embodiment of the present invention, the material of the multitude of porous elements is a polymeric material.

According to an embodiment of the present invention, the material of the multitude of porous elements is a polymeric material with a conversion of >50% and <100%.

In the sense of the present invention, the term "conversion" especially includes, means or refers to a measurement according to the following procedure:

After the polymerization of the material of the multitude of porous elements the, phase separated liquid as well as remaining monomers and initiators were removed by washing with a suitable solvent After that, the sample was blow-

dried until there was no change in weight anymore. The conversion was calculated as follows: conversion = P/M _o * 100 %

where P is the weight of the dry copolymer composite network obtained from the sample and M ₀ is the weight of the monomers in the feed.

According to an embodiment of the present invention, the material of the multitude of porous elements is a polymeric material with a conversion of >70% and <95%.

The term "essentially" means and/or includes especially a wt-% content of >90 %, according to an embodiment >95 %, according to an embodiment >99 %.

According to an embodiment of the present invention, the material of the multitude of porous elements is a polymeric material comprises a poly (methy)acrylic material.

According to an embodiment of the present invention, the porous element comprises a poly(meth)acrylic material made out of the polymerization at least one polyfunctional (meth)acrylic monomer. According to an embodiment of the present invention, the polyfunctional (meth)acrylic monomer is a bis-(meth)acryl and/or a tri-(meth)acryl and/or a tetra-(meth)acryl and/or a penta-(meth)acryl monomer.

According to an embodiment of the present invention, the polyfunctional (meth)acrylic monomer is chosen out of the group comprising bis(meth)acrylamide, tripropyleneglycol di(meth)acrylates, pentaerythritol tri(meth)acrylate, tetraethyleneglycoldimetacrylate, polyethyleneglycoldi(meth)acrylate, ethoxylated bisphenol-A-di(meth)acrylate or mixtures thereof.

According to an embodiment of the present invention, the porous element comprises a poly(meth)acrylic material made out of the polymerization at least one polyfunctional (meth)acrylic monomer and at least one (meth)acrylic

monomer.

According to an embodiment of the present invention, the (meth)acrylic monomer is chosen out of the group comprising (meth)acrylamide, (meth)acrylic acid, hydroxyethyl(meth)acrylate, ethoxyethoxyethyl(meth)acrylate hydroxyethylmeth(meth)acrylate, isobornyl(meth)acrylate, isobornyl meth(meth)acrylate, glycidyl methacrylate, N-hydroxy succinimide (meth) acrylate or mixtures thereof.

According to an embodiment of the present invention, the crosslink density in the poly(meth)acrylic material is >0.05 and <1. In the sense of the present invention, the term "crosslink density" means or includes especially the following definition: The crosslink density δ _x is

here defined as δ „ = where X is the mole fraction of polyfunctional x L + X monomers and L the mole fraction of linear chain (= non polyfunctional) forming monomers. In a linear polymer δ _x = 0 , in a fully crosslinked system δ _x = 1 . According to a further embodiment, the device comprises at least one adhesion promoting layer between the multitude of porous elements and the substrate.

According to a preferred embodiment of the present invention, the adhesion promoting layer is a monolayer. Preferably the at least one adhesion promoting layer is chosen from the group silane-containing layers, thiol-containing layers, amine-containing layers or mixtures thereof.

The term "silane-containing layer" especially means and/or includes a layer which comprises a material of the form

whereby Ri is selected out of the group comprising acrylate, methacrylate,

acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol;

R-2 is selected out of the group alkylene, arylene, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or poly disulfides, R-3 and R ₄ are independently selected out of the group halogen, R6-R7

(whereby R ₆ is selected out of the group comprising acrylate, methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol and R7 is selected out of the group alkyl, aryl, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or polydisulfides), O-Rg (whereby R ₈ is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, heteroaryl, halogen)

R ₅ represents the group 0-R ₉ , where R ₉ is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, halogen and/or R ₅ is a hydrolyzable moiety.

Generic group definition: Throughout the description and claims generic groups have been used, for example alkyl, alkoxy, aryl. Unless otherwise specified the following are preferred groups that may be applied to generic groups found within compounds disclosed herein: alkyl: linear and branched Cl-C8-alkyl, alkylene: selected from the group consisting of: methylene; 1,1 -ethylene; 1,2-ethylene; 1,1-propylidene; 1,2- propylene; 1,3- propylene; 2,2-propylidene; butan-2-ol-l,4-diyl; propan-2-ol-l,3- diyl; 1, 4-butylene; cyclohexane-l,l-diyl; cyclohexan-l,2-diyl; cyclohexan-1,3- diyl; cyclohexan-l,4-diyl; cyclopentane- 1,1 -diyl; cyclopentan-l,2-diyl; and cyclopentan- 1,3-diyl, long-chain alkyl: linear and branched C5-C20 alkyl alkenyl: C2-C6-alkenyl, cycloalkyl: C3-C8-cycloalkyl, alkoxy: Cl-C6-alkoxy, long-chain alkoxy: linear and branched C5-C20 alkoxy aryl: selected from homoaromatic compounds having a molecular weight under 300,

arylene: selected from the group consisting of: 1 ,2-phenylene; 1,3- phenylene; 1,4-phenylene; 1,2-naphtalenylene; 1,3-naphtalenylene; 1,4- naphtalenylene; 2,3-naphtalenylene; l-hydroxy-2,3-phenylene; l-hydroxy-2,4- phenylene; l-hydroxy-2,5- phenylene; and l-hydroxy-2,6-phenylene, heteroaryl: selected from the group consisting of: pyridinyl; pyrimidinyl; pyrazinyl; triazolyl; pyridazinyl; 1,3,5-triazinyl; quinolinyl; isoquinolinyl; quinoxalinyl; imidazolyl; pyrazolyl; benzimidazolyl; thiazolyl; oxazolidinyl; pyrrolyl; carbazolyl; indolyl; and isoindolyl, wherein the heteroaryl may be connected to the compound via any atom in the ring of the selected heteroaryl, amine: the group -N(R)2 wherein each R is independently selected from: hydrogen; Cl-C6-alkyl; Cl-C6-alkyl-C6H5; and phenyl, wherein when both R are Cl-C6-alkyl both R together may form an - NC3 to an -NC5 heterocyclic ring with any remaining alkyl chain forming an alkyl substituent to the heterocyclic ring, halogen: selected from the group consisting of: F; Cl; Br and I, polyether: chosen from the group comprising-(O-CH ₂ -CH(R)) _n -OH and -(O-CH ₂ -CH(R)) _n -H whereby R is independently selected from: hydrogen, alkyl, aryl, halogen and n is from 1 to 250

Unless otherwise specified the following are more preferred group restrictions that may be applied to groups found within compounds disclosed herein: alkyl: linear and branched Cl-C6-alkyl, long-chain alkyl: linear and branched C5-C10 alkyl, preferably linear

C6-C8 alkyl alkenyl: C3-C6-alkenyl, cycloalkyl: C6-C8-cycloalkyl, alkoxy: Cl-C4-alkoxy,

long-chain alkoxy: linear and branched C5-C10 alkoxy, preferably linear C6-C8 alkoxy aryl: selected from group consisting of: phenyl; biphenyl; naphthalenyl; anthracenyl; and phenanthrenyl, heteroaryl: selected from the group consisting of:

pyridinyl; pyrimidinyl; quinolinyl; pyrazolyl; triazolyl; isoquinolinyl; imidazolyl; and oxazolidinyl, wherein the heteroaryl may be connected to the compound via any atom in the ring of the selected heteroaryl, heteroarylene: selected from the group consisting of: pyridin 2,3-diyl; pyridin-2,4-diyl; pyridin-2,6- diyl; pyridin-3,5-diyl; quinolin-2,3-diyl; quinolin-2,4-diyl; isoquinolin-l,3-diyl; isoquinolin-l,4-diyl; pyrazol-3,5-diyl; and imidazole-2,4-diyl, amine: the group -N (R) 2, wherein each R is independently selected from: hydrogen; Cl-C6-alkyl; and benzyl, halogen: selected from the group consisting of: F and Cl, polyether: chosen from the group comprising-(O-CH ₂ -CH(R)) _n -OH and -(O-CH ₂ -CH(R)) _n -H whereby R is independently selected from: hydrogen, methyl, halogen and n is from 5 to 50, preferably 10 to 25.

It has been shown for a wide range of applications that this silane- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the microfluidic bio-separation device.

The term "thiol-containing layer" especially means and/or includes a layer which comprises a material of the form R-SH with R chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl. It has been shown for a wide range of applications that this thiol- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If a thiol- containing layer is used, the surface of the matrix material is chosen out of a thiol- binding material, especially the surface of the matrix material is an Au-surface. The term "amine-containing layer" especially means and/or includes a layer which comprises a material of the form Ri-NH-R ₂ with Ri chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether and R ₂ chosen out of the group hydrogen, alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether.

It has been shown for a wide range of applications that this amine- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If an amine-

containing layer is used, the surface of the matrix material is preferably equipped with amine-binding groups, preferably epoxy groups and/ or reactive esters, halogenides and/or amines.

The present invention furthermore relates to a method of making a multitude of porous elements, especially as described within this application comprising a (photo-) polymerization step.

The present invention furthermore relates to a method of making a multitude of porous elements, especially as described within this application comprising a (photo-) polymerization induced phase separation (PIPS). According to an embodiment of the present invention, the method of making a substrate material according to the present invention comprises the following steps: a) Filling, preferably completely filling a preferably commercially available substrate with a photo -polymerizable fluid comprising suitable monomers and at least one non-polymerizable liquid material (in the context of this application to be called "solvent"), b) subsequently locally photo -polymerizing c) a washing step to remove the non-cross linked fluid (e.g. the "solvent" and non-reacted monomers). Preferably the solvent comprises a material selected out of the group comprising alkyl-substituted aryl components, especially xylene, toluene etc.; decaline, tetrahydronaphtalene; N-methylpyrolidone; glycerol; glycols and polyglycoles, especially tripropyleneglycol, polypropyleneglycole, diethyleneglycol, triethyleneglycol . tetraethyleneglycol Preferably the wt% ratio of monomer: solvent is >2: 1 and <1 :20.

According to an embodiment of the present invention, step (a) is performed involving capillary forces, suction forces, centrifugal forces, doctor blading, spin coating and/or dipping.

According to an embodiment of the present invention, step (b) is performed using a mask, especially a lithographic mask. However, according to an alternative and/or additional embodiment of the present invention, step (a) is

performed involving a substrate patterned (prior to step a) with an array of individual wetting areas surrounded with a grid of non-wetting areas. After coating of a thin film of photo-polymerizable fluid, spontaneously the layer breaks up in an array of small individual droplets. A substrate material and/or a material made according to a method according to the present invention may be of use in a broad variety of systems and/or applications, amongst them one or more of the following: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures such as e.g. blood or saliva high throughput screening devices for chemistry, pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e.g. in criminology, for on- site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics tools for combinatorial chemistry analysis devices - high performance liquid chromatography (HPLC), monolithic column chromatography, capillary electrophoresis chromatography (CEC), and reverse phase (RP) chromatography size exclusion, adsorption, ion exchange or affinity chromatography integrated part of micro fluidic biosensors for pre-amplifϊcation (e.g. before PCR), filtering or detection, substrate for improved Boom method for DNA collection/ purification rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures (e.g. blood, saliva), for on-site testing and for diagnostics in centralized laboratories - applications are in medical (DNA/protein diagnostics for cardiology, infectious disease and oncology), food, and environmental diagnostics,

metabolomics

The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, the figures and the following description of the respective figures and examples, which — in an exemplary fashion — show several preferred embodiments of a substrate material as well as a device according to the invention.

Fig. 1 shows a substrate according to Example I with a thin layer of water applied to show the patterning of the substrate

Fig. 2 shows a top view of a plurality of porous elements according to a first embodiment of the present invention;

Fig. 3 shows a detailed view of one of the porous elements of Fig.

2; and

Fig. 4 is an enlarged view of Fig. 3 showing the surface structure of the porous element of Fig. 3

Figs. 1 to 4 refer to Example I of the present invention in which - in a merely illustrative way - one example of a substrate and a multitude of porous elements is shown.

Example I was made as follows.

A glass substrate 50x50mm was and cleaned with Merck Extran MA 02 soap, rinsed with dematerialized water and blow dried. Afterwards it was treated with UV ozone for 10 minutes.

A first layer of a HPR 504 positive resist was brought on the substrate via Spin coating (lOOOprm), after that the substrate was baked on hotplate at 90°C for 5 minutes.

A mask pattern was brought up on the substrate, using as exposure tool a Thermo Oriel IOOOW (3 mW/cm ² , 40 sec). The pattern was developed in a PLSI developer for 90 sec. UV ozone treatment for 10 minutes followed, then a

1H,1H,2H,2H-Perfluorodecyltrichlorosilane (ABCR) deposition in dessicator (<lmBar) for 30 minutes. The photoresist was removed with acetone, then the substrate was rinsed with isopropanol.

Fig. 1 shows (merely for illustrative reasons) the picture after applying a thin layer of water on the resulting sample. It can be clearly seen that the layer spontaneously broke up in small individual droplets wetting the hydrophilic areas.

After the treatment of the substrate, the porous elements were made the following way: A monomer mixture (50% TPG solvent with 48% TEGDMA and

2% photoinitiator IRG 651) was brought up by doctor blading to form a layer of approx. 50μm thickness. It should be noted that this is just one possible alternative, spin coating, plotting or ink jet printing also give good results.

Subsequently the material was UV photopolymerized (lOmin flood exposure) in a N2 protective gas environment.

After that step the sample was rinsed in ethanol for about 5 min and blow-dried. Fig. 2 shows the resulting array of porous elements.

As can be seen in Fig. 3 the single porous elements have a somewhat "finger hut pillar" shape with a good open porosity (as follows from Fig. 4).

The particular combinations of elements and features in the above

detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.