The present invention relates to novel methods for producing filter
elements which comprise a microporous element. Such filter elements are commonly
used for micro filtration, e.g., for solid-liquid separation, clearing of solutions,
harvesting bacteria, and the like. Filter elements are also used for column chromatography,
adsorption/immobilisation of proteins, e.g. biocatalysts, as retainer for granular
There is an increasing demand in the area of analytical chemistry
for devices capable of handling moderate to small sample volumes, in a manner which
is rapid, gives high recovery and minimizes any possibility of sample contamination.
Among other desired attributes are low cost, ease of manufacture and suitability
for application with conventional equipment.
Previous devices for micro filtration etc. involve the use of preformed
filter material. Synthetic polymers such as nylon, methacrylate or semisynthetic
polymers such as nitro cellulose, or cellulose acetate have been used over decades
for preparation of microporous membranes. Mostly the filter material is a nonwoven
material which is formed from a web of synthetic or natural fibers. The fibers
may or may not be bonded together by a binder. In general, discs are cut from the
nonwoven material and positioned within a sample tube or the like. Problems in
connection with this previous approach include an insufficient contact of the pre-cut
filter disc to the wall of the filter tube leaving small gaps and thereby allowing
the applied liquid sample to escape.
If the filter disc is attached to the filter tube by an adhesive,
the adhesive may influence the filter properties of the material in an uncontrollable
manner. The same holds true when the filter disc is welded into the filter tube
by locally applying heat or by ultrasonic treatment.
The problems discussed above are enhanced when the overall size of
the filter element is reduced. It has therefore hitherto not been technically feasible
to produce satisfactory filter elements with apertures of a diameter as low as
It is therefore an object of the present invention to provide a cost-effective
method for producing a filter element whereby a complete circumferential contact
of the microporous element to the support is readily obtained even with a small
In its broadest aspect, the present invention relates to a method
for producing a filter element by generating a microporous element within an aperture
of a solid moisture-impervious support, comprising the steps of applying a liquid
phase to the aperture so as to form a self-sustaining liquid layer over the cross-section
of the aperture; and causing solidification in spongy form of at least part of
the liquid phase.
Accordingly, the microporous element is generated in situ from a liquid
precursor. The liquid precursor readily takes a shape that matches the shape of
the aperture in the support. Any imperfections like burrs etc. of the aperture
are hereby compensated for. Upon solidification, a microporous element is obtained,
which snugly fits into the aperture of the support and has complete circumferential
contact to the walls thereof.
Preferably, the support is formed of a plastic such as polypropylene,
polyethylene, propylene/ethylene copolymer, polyvinyl acetate, polyamide, polystyrene,
polyethylene terephthalate, polyether etherketon (PEEK), polycarbonate, polyethylene
vinylacetate, polyester, polyimide, or mixtures thereof. Also included are composite
materials of plastic with fibres or frames of glass, silicon dioxide, carbon or
Usually, the support has the form of a tube, preferably with circular
cross-section, and the microporous element is generated at or near to one edge
of the tube. In order to facilitate sample application and for accommodation of
greater sample volumes, preferably at least a section of the tube is of conical
form, having a smaller and a larger cross-sectional end, with the microporous element
generated at or near to the smaller cross-sectional end. For example, the support
is a pipette tip such as commonly used with Eppendorf pipettes. In order to prevent
dislocation, such as slipping, of the microporous element formed, the tube may
have a structured inner surface, like a surface with rings or grooves.
It is also envisaged to arrange a plurality of supports, e.g., up
to several hundreds, in parallel alignment to form a multiple channel filter element.
The multiple channel filter element will allow a biological sample to be tested
simultaneously against hundreds of reagents. Alternatively, the support can comprise
a plurality of apertures, e.g., in the form of parallel bores or tapered holes.
When the multiple channel element is to be evaluated optically, it is convenient
to include an opacifying agent such as carbon black into the support material to
prevent interference from neighbouring channels.
There are several possibilities of applying the liquid phase to the
aperture of the support. The various methods will be exemplified with reference
to a tubular support but will not be limited thereto. As the liquid phase in general
is a solution the terms "liquid phase" and "solution" will be used interchangeably
unless otherwise required by the context. Conveniently, application of the solution
to the aperture is accomplished by allowing the solution to ascend in the tube
by capillary action. The support, for example a tube, is dipped into the solution
and raised again. Due to the surface tension of the solution, a liquid layer will
remain in the aperture over the cross-section of the aperture. If, for example,
the viscosity of the solution is too high different methods for applying the solution
to the aperture may be adopted. The ascending force of the solution may be enhanced
by temporarily sealing the distant end of the tube, slightly heating the tube,
then dipping the tube with its free end into the solution, and allowing the tube
to cool to ambient temperature, whereby the solution is drawn into the tube by
the volume contraction of the enclosed air. Alternatively, the solution can be
introduced from the distant end of the tube and may be brought into its final position
by centrifugation. Preferably, the end at or near to which the microporous element
is to be formed is sealed with a cap or by pressing against an elastic plate. The
cap or the elastic plate preferably has microgrooves or micropores for allowing
the enclosed air to escape. Then the arrangement is subjected to centrifugation
during which the solution migrates to the end of the tube where it is retained
at least partially by the cap or elastic plate. The cap or the elastic plate may
be removed before or after solidification, in particular before solidification
of the solution or of a part of the solution.
The practitioner often faces the problem that aqueous samples that
are introduced in containers made of hydrophobic material tend to adhere to the
wall in drops rather than flowing down and collecting at the bottom. In order to
avoid this phenomena, the inner wall of a tube which acts as support for a filter
element according to the invention, may be coated with a hydrophilic coating. The
hydrophilic coating will prevent aqueous samples from adhering to the wall of the
support tube. Also, there is no adsorptive loss of biopolymers due to adsorption
to the wall of the support. If the inner wall of the tube is coated with the hydrophilic
coating, the edge of the tube next to the microporous element is preferably kept
free of hydrophilic coating. This will prevent sample liquids which exit from the
edge of the tube next to the microporous element from creeping to adjacent filter
elements, especially in the embodiment of the present invention where a plurality
of filter elements is arranged to form a multiple channel filter element.
The hydrophilic coating is conveniently prepared by applying a solution
of one or more polyvinyl esters in an organic solvent to the inner wall of the
tube, allowing the organic solvent to evaporate, and partially hydrolyzing the
resulting layer of polyvinyl ester at the surface thereof. Suitable polyvinyl esters
include polyvinyl acetate (a molecular weight of about 500000 is generally suitable),
polyvinyl propionate and polyvinyl stearate.
Partial hydrolysis of the layer of polyvinyl ester is performed by
contacting the layer of polyvinyl ester with an alkaline aqueous solution, such
as sodium hydroxide. The edge of the tube next to the microporous element can be
kept free of hydrophilic coating by applying the solution of polyvinyl ester only
to a part of the inner wall of the tube, for example by partially immersing the
tube into the solution of polyvinyl ester. After hydrolysis, the microporous element
can be generated at or near to the edge of the tube, that has not been brought
into contact with the polyvinyl ester solution. Alternatively, the entire inner
wall of the tube can be coated with polyvinyl ester, but only part thereof is hydrolyzed.
Alternatively, the hydrophilic coating can be generated by using a
high molecular weight polypropylene glycol, for example having a molecular weight
of 4000 or higher. Such polypropylene glycols show moderate to good solubility
in cold water, however poor solubility in warm water. Accordingly, a cold aqueous
solution of polypropylene glycol, for example at 0░ to 4░C, might be introduced
into the tube and subsequently the temperature is raised, for example to about
20░C. The polypropylene glycol coming out of the solution shows a high affinity
to the inner wall of the tube and deposits thereon as a thin layer. Excess polypropylene
glycol solution is then removed. Additional chemical crosslinking may be advantageous
in some cases.
Employing the method(s) according to the present invention, filter
elements having an aperture of a diameter from 0.02 mm to 4 mm, in particular from
0.2 to 2.0 mm, can be produced.
In a first embodiment, the present invention relates to a method for
producing a filter element by generating a microporous element within an aperture
of a solid moisture-impervious support, comprising the steps of providing a solution
of a synthetic or semi-synthetic resin in a solvent; applying the solution to the
aperture so as to form a self-sustaining liquid layer over the cross-section of
the aperture; and causing a nonsolvent to diffuse into the layer, which nonsolvent
is miscible with the solvent, whereby the resin precipitates to form the microporous
Preferably the resin is selected from the group consisting of polyvinyl
esters, partially deacylated polyvinyl esters, cellulose derivatives, polyamides,
and mixtures thereof. Among polyvinyl esters polyvinyl acetate, polyvinyl propionate,
polyvinyl stearate, and polyvinyl cinnamic acid ester; among cellulose derivatives
nitrocellulose, and cellulose propionate are to be mentioned. A suitable polyamide
is Nylon 6/6.
In certain instances, the resin preferably comprises both hydrophilic
and hydrophobic segments within its molecules. Suitable resins include poly(vinyl
alcohol-co-ethylene), poly(vinyl alcohol-co-vinylacetate), ethylene acrylic acid
copolymer, ethylene acrylic ester copolymer, ethylene acrylamide copolymer, acrylic
acid vinylacetate copolymer, acrylamide vinylacetate copolymer, copolymer of acrylic
acid diamine monoamide with vinylacetate, poly(vinyl alcohol-co-styrene), acrylamide
acrylic ester copolymer, and mixtures thereof. Specifically, copolymers of acrylamide
with hexyl acrylate, propyl acrylate or dodecyl acrylate are useful.
Preferably the solvent is selected from dimethyl sulfoxide, dimethylformamide,
dimethylacetamide, formamide, formic acid, acetic acid, 2,2,2-trichloro ethanol,
and mixtures thereof.
Preferably the nonsolvent is selected from water, alcohols having
1 to 4 carbon atoms, ammonia, ethylacetate, acetone, ethylenediamine, and mixtures
thereof. The nonsolvent may be either liquid or gaseous.
The nonsolvent is caused to diffuse into the liquid layer of the resin
solution by various methods. A liquid nonsolvent may be brought into contact with
the liquid layer of resin solution from one or both sides thereof, for example
by dipping the end of the tube at which the layer of resin solution is positioned
into a liquid nonsolvent. Additionally or alternatively, nonsolvent may be introduced
from the distant end of the tube. If a gaseous nonsolvent is to be used, the arrangement
of support with layer of resin solution is positioned within an atmosphere which
is saturated or nearly saturated with the vapours of the nonsolvent. Precipitation
may also be accomplished in two successive steps by firstly applying gaseous nonsolvent
and, after partial solidification, subsequently applying liquid nonsolvent.
Plane upper and lower surfaces of the microporous element are obtained
when the final concentration of nonsolvent in the resin solution during precipitation
is raised to about 50% by weight. A plane upper surface allows for a more uniform
filtration performance of the filter element. A plane lower surface provides even
contact with, e.g., blotting membranes onto which an adsorbed material is to be
Especially favourable results are obtained when one of the following
combinations of resin/solvent/nonsolvent is used: one of poly(vinylalcohol-co-ethylene),
nitrocellulose, cellulose propionate, or polyvinylacetate as resin, dimethyl sulfoxide
as solvent and water as nonsolvent; or polyamides (like Nylon 6,6) as resin, 2,2,2-trichloro
ethanol as solvent and acetone as nonsolvent.
Without intending to be bound to theory it is believed that in generating
the microporous element according to this first embodiment of the present invention
the following mechanisms are involved: When the nonsolvent diffuses into the layer
of resin solution, the solubility of the resin is gradually decreased. As the limit
of solubility is reached the resin begins to precipitate from the solution at individual
points. The precipitation of the resin proceeds at the points of initial precipitation.
Ultimately, the solvent/nonsolvent is enclosed in large interconnecting enclaves
in a solid matrix of resin. The interconnecting enclaves form the liquid-permeable
channels of the final microporous element. If a synthetic resin is used which
comprises both hydrophilic and hydrophobic segments, the hydrophobic segments will
be forced towards each other and brought into contact with each other as the concentration
of nonsolvent in the resin solution increases. There will be interactions between
the hydrophobic segments of neighbouring molecule chains, which result in the formation
of a crystalline hydrophobic backbone of the precipitated resin. The hydrophilic
segments will be oriented towards the enclaves filled with solvent/nonsolvent.
Accordingly, a microporous element is obtained where the liquid-permeable channels
are predominantly hydrophilic. This provides the benefit of biocompatibility. The
term "biocompatibility" means that the three-dimensional structure of biopolymers,
for example proteins, is maintained. The interphase forces are less destructive
when the polymer surface is rich in hydroxyl, amide or ether groups.
In order to modify the adsorptive properties of the microporous element,
the solution of the synthetic resin may further comprise solid microparticles.
The micro particles may be composed of silicon dioxide, silica gel, aluminium oxide,
titan dioxide, zirconium oxide, glass, carbon or graphite. Also, the particles
can be composed of inorganic material, such as calcium phosphate, zinc polyphosphate
or the like. Another type of granular microparticles consists of an inorganic core
such as microporous silicagel with a microlayer of organic polymer. The pores and
the surface of the grain may be modified in a way, that macromolecules are restricted
from penetrating into the pores ("Restricted access material"). Also, the micro
particles may consist of organic material such as a powder of cured resin, or highly
crosslinked polysaccharides, as are available under the sephadex tradename, however
care has to be taken in selecting an organic material in that it must not be soluble
in the solvent used. The particles can be non-porous or porous, but preferably
are porous with a preferred pore size in the range of 1 nm to 500nm. Generally,
the particles have a size from 5 nm to 80 Ám, in particular from 0.5 Ám to 30 Ám,
however porous microparticles preferably have a size of 1 Ám or more, whereas non-porous
microparticles preferably have a size of 1 Ám or less. The microparticles can be
pretreated, e.g., derivatized, such that the adsorbent properties thereof meet
specific requirements. Any kind of commercially available adsorbent particles as
used for solid phase extraction or chromatography, such as affinity chromatography
with proteins, antibodies, peptides, carbohydrates, nucleic acids, or for ion exchange
chromatography, immuno chromatography, hydrophobic interaction chromatography,
chelating chromatography and reversed phase chromatography are useful. Materials
suited for high performance liquid chromatography are especially useful. For example
proteins, such as specific antibodies, lectins, avidin, receptor-proteins, enzymes,
synthetic peptides, nucleic acids or oligonucleotides may be attached to the microparticles,
either covalently or via linkers. The adsorbent particles have a granular shape,
for example spherical. The microparticles may be used in an amount of up to 50mg,
preferably 100ng to 20mg, per filter element.
In the final microparticle-containing filter element, the outer or
inner surfaces of the enclosed microparticles are accessible to an applied liquid
sample, and adsorption/immobilization of analytes contained in the liquid sample
can take place.
In a second embodiment, the invention relates to a method for producing
a filter element by generating a microporous element within an aperture of a solid
moisture-impervious support, comprising the steps of providing an aqueous solution
of a hydrocolloid, which comprises solid microparticles slurried therein; applying
the solution to the aperture so as to form a self-sustaining liquid layer over
the cross-section of the aperture; causing the hydrocolloid solution to solidify;
and, optionally, one or both of desiccating and crosslinking said layer of the
solidified hydrocolloid solution.
Preferably, the hydrocolloid is selected from low melting agarose,
starch, polyvinyl alcohol, and mixtures thereof. The aqueous solution of the hydrocolloid
contains preferably 1 to 10% by weight, in particular 2 to 5% by weight of the
solution, of the hydrocolloid. When polyvinyl alcohol is used as the hydrocolloid,
the addition of up to 0,2% by weight of the solution, of sodium tetraborate or
of up to 50% by weight of the solution, of dimethyl sulfoxide is sometimes advantageous.
These hydrocolloids are poorly soluble in cold water, however disperse
or dissolve upon heating. Preferably, a hot hydrocolloid solution is applied to
the aperture of the carrier. Upon cooling, the hydrocolloid solution solidifies.
In one alternative, the solidified hydrocolloid solution is subsequently
desiccated. Desiccation can be accelerated by heating the arrangement to a temperature
of about 40░C. Alternatively, in particular when using heat sensitive material,
desiccation can be achieved by placing the arrangement in a closed chamber over
a desiccating agent such as phosphorus pentoxide. The final moisture content preferably
is less than 1 mbar water vapor partial pressure.
Upon desiccation, the solidified layer of hydrocolloid solution shrinks.
In absence of the microparticles mentioned above, the solidified layer of hydrocolloid
solution would shrink away from the inner wall of the support with the effect that
no useful filter element would be obtained. According to the invention, microparticles
are provided in the aqueous solution of hydrocolloid which act as pore-forming
agent during desiccation. Accordingly, complete circumferential contact of the
layer of hydrocolloid solution to the wall of the support is maintained and a plurality
of microscopic cracks between the microparticles are formed upon desiccation. Also,
the microparticles act so as to control or modify the adsorptive properties of
the layer thus obtained. The microscopic cracks act as the liquid-permeable channels
of the final microporous element. A proportion of 5 to 50%, calculated by weight
of hydrocolloid solution, of microparticles will generally be useful.
The obtained layer of solidified hydrocolloid can be subjected to
crosslinking instead of or after desiccation. For this purpose the microporous
element is treated with crosslinking agents, e.g., boric acid, sodium tetraborate,
phosphorus oxide chloride, epichlorhydrin or bisoxiranes, such as 1,4-butanediol
diglycidyl ether. Polyvinylalcohol can be crosslinked with sodium tetraborate by
applying alkaline pH for gelation. Both desiccation and crosslinking have the effect
of increasing the mechanical stability and solvent-resistance of the final microporous
In a preferred method, an aqueous hydrocolloid solution, in particular
an aqueous solution of low melting agarose, which comprises solid microparticles
slurried therein is applied to the aperture of a solid moisture-impervious support
so as to form a self-sustaining liquid layer over the cross-section of the aperture,
then a channel-sparing nonsolvent is permeated through the liquid layer while retaining
the layer by means of a retaining tool as explained in detail below, then the retaining
tool is removed and the hydrocolloid solution is caused to solidify. Finally, the
solidified hydrocolloid layer is preferably subjected to crosslinking.
Useful microparticles are those discussed above in connection with
the first embodiment of the invention. Additionally, microparticles coated with
dextran, such as dextran-coated charcoal, dextran-coated poly(styrene-divinyl benzene)
or biological structures absorbed to a porous support, such as ribosomes, nucleosomes,
chromosomes, synaptosomes, phages, plasmids, all preadsorbed to porous or non-porous
supporting beads, are to be mentioned. The microparticles preferably have a size
from 0.5 Ám to 30 Ám.
In a third embodiment, the present invention relates to a method for
producing a filter element by generating a microporous element within an aperture
of a solid moisture-impervious support, comprising the steps of providing a solution
of a monomer or a mixture of monomers in a solvent, optionally comprising crosslinking
monomers, applying the solution to the aperture so as to form a self-sustaining
liquid layer over the cross-section of the aperture; and causing the monomer(s)
Preferably, the monomers are ethylenically unsaturated monomers such
as vinylacetate or other vinylesters, acrylic acid and its derivatives. Preferred
acrylic acid derivatives include acrylic acid amides, such as acrylamide, N,N-dimethyl
acrylamide, acrylic acid diamine monoamide; acrylic esters, such as butyl acrylate,
dodecyl acrylate, octadecyl acrylate, vinyl acrylate, 2,3-epoxypropyl acrylate,
diethylaminoethyl acrylate, 2-dimethylaminoethyl acrylate, and 3-sulfopropyl acrylate.
Crosslinking monomers are monomers having two or more sites of ethylenical
unsaturation. A preferred crosslinking monomer is N,N'-methylenebis(acrylamide).
The presence of crosslinking monomers increases the mechanical stability and solvent-resistance
of the final microporous element.
As a solvent water, dimethyl sulfoxide, dimethylformamide, dimethylacetamide,
formamide, formic acid, acetic acid, 2,2,2-trichloro ethanol, or mixtures thereof
are useful, depending on the nature of the monomer(s) used.
In a preferred method, an aqueous solution of acrylic acid or its
derivatives, preferably together with crosslinking monomers, which comprises solid
microparticles slurried therein is applied to the aperture of a solid moisture-impervious
support so as to form a self-sustaining liquid layer over the cross-section of
the aperture, then a channel-sparing nonsolvent is permeated through the liquid
layer while retaining the layer by means of a retaining tool as explained in detail
below, then the retaining tool is removed and the acrylic acid (derivatives) caused
A proportion of 1 to 10% by weight of crosslinking monomer(s), for
example N,N'-methylenebis(acrylamide), calculated on the total monomer, is generally
Also a mixture of hydrophilic and hydrophobic monomers is useful.
In this case, there will be interactions between the polymerized units of the hydrophobic
monomers of the growing molecule chains, which result in the formation of a crystalline
hydrophobic backbone of the polymer formed. The units of the hydrophilic monomers
will be orientated towards the enclaves filled with the solvent. Accordingly, a
microporous element is obtained where the liquid-permeable channels are predominantly
Sometimes the monomer or mixture of monomers is soluble in the solvent,
whereas the growing polymer becomes increasingly insoluble in the selected solvent
and finally precipitates from the solution. A mixture of solvents may be useful
in some instances. A nonsolvent can be caused to diffuse into the layer of polymer
solution after polymerization, in order to precipitate the polymer formed or to
complete precipitation of the polymer formed. Also, a porogenic liquid, such as
n-dodecanol or polypropylene oxides, can be present during polymerisation. The
polymerisation may also result in a polymer gel swelled with the solvent used.
Polymerisation of the ethylenically unsaturated monomers is generally
effected under the influence of free radicals. Free radicals are generated from
the usual thermally decomposable initiators or a combination of free radical initiator
and reducing agent. Usually, the initiator is added to the monomer solution. The
concentration of the initiator is adjusted to provide sufficient pot life for application
of the monomer solution to the aperture of the support. A combination of N,N'-tetramethyl
ethylene diamine and a persulfate is especially preferred as initiator. In the
alternative, an aqueous solution of initiator or initiator/reducing agent is used
which is in contact by one or both sides of the liquid layer of monomer solution.
Also photoinitiators such as riboflavin are useful for initiating the polymerization.
Instead of ethylenically unsaturated monomers, a monomer or mixture
of monomers can be used which are capable of undergoing polyaddition or polycondensation.
Among useful monomers are a combination of diamines with polyepoxides, such as
a combination of ethylenediamine with 1,4-butanediol diglycidylether. Further
examples include polypropylene oxide diamine and 1,4-butanediol, each with 1,4-butanediol
This third embodiment involves the benefit of a lower viscosity of
the monomer solution compared to a solution of a polymer of the same monomers at
a corresponding concentration. The lower viscosity facilitates application of the
solution to the aperture of the support.
In order to modify the adsorptive properties of the microporous element,
the monomer solution may further comprise solid microparticles. Microparticles
that are useful, have been discussed above in connection with the first embodiment
of the invention.
Where microparticles are used in the first or the third embodiment
as well as in the second embodiment, it is sometimes advantageous to permeate the
microparticle-containing liquid layer in the aperture of the support with a channel-sparing
non-solvent in order to displace excessive hydrocolloid solution, resin solution
or monomer solution, respectively. The channel-sparing nonsolvent must be non-miscible
with the solvents used. Permeation can be effected by pressing or drawing channel-sparing
nonsolvent through the microparticle-containing layer whilst retaining the layer
by means of a retaining tool consisting of a microporous filter. The hydrocolloid
solution, resin solution or the monomer solution, respectively, is displaced from
the larger interstices between the microparticles and accumulates at the points
of contact of the microparticles. The channel-sparing nonsolvent keeps the interstices
free, thus producing channels. The retaining tool is removed before solidification
of the hydrocolloid or polymerisation of the monomer(s) or precipitation of the
resin. Upon solidification/polymerisation/precipitation, the microparticles are
linked one to another at their points of contact. The channel-sparing nonsolvent
is removed by washing with a suitable liquid. As channel-sparing nonsolvent, silicon
oil is preferred.
Also possible are combinations of two or more of the three embodiments
discussed above. For example, a microparticle-containing layer could be prepared
according to the third embodiment and subsequently be impregnated with a resin
solution, in which resin is precipitated according to the first embodiment.
In the various embodiments of the invention, the handling of very
small liquid volumes is involved. Undesired evaporation of solvents before solidification
can be avoided when all operations are performed in closed systems with solvent-saturated
atmosphere. Preferably the temperature is precisely controlled.
The invention further relates to the filter elements which are obtainable
by the various embodiments discussed above. The filter elements according to the
invention can be applied for most versions of analytical or micro preparative liquid
chromatography such as affinity chromatography, immuno chromatography as well as
for binding studies enabling also the isolation of multi component binding complexes.
The filter elements may be especially useful in biotechnology, in
molecular biology and in medical biochemical diagnostics, allowing low-cost screening
of hundreds of samples in parallel.
The following advantages were achieved: The method opens the way for
far going miniaturization, avoids unspecific loss of biopolymer by presenting biocompatible
surfaces and by avoiding the use of frits, saves cost by using expensive microparticle
material in very small amounts. In addition transfer of separated substances from
the filter to blotting membranes is possible without having problems with dead
volumes. Production at low cost allows the filter to be used only once, avoiding
the risk of contamination. The possibility of using microparticles with known or
standardized adsorptive properties allows for the manufacture of filter elements
with predictable adsorption characteristics and facilitates quality control.
The present invention will be described in more detail below with
reference to the exemplary embodiments which are schematically illustrated in the
following drawings, in which:
- Figure 1 shows a filter element obtained according to a preferred embodiment
of the invention; and
- Figure 2 illustrates the mode of operation of the channel-sparing nonsolvent.
Figure 1 shows a filter element obtained according to a preferred
embodiment of the invention. The filter element comprises a moisture-impervious
support (1) having a hydrophilic coating (2) at the inner wall thereof. The filter
element further comprises microparticles (3) in a matrix (4) of precipitated resin,
solidified hydrocolloid or polymerized monomer.
Figure 2 illustrates the mode of operation of the channel-sparing
nonsolvent. The channel-sparing nonsolvent (6), shown in cross-section, keeps the
larger interstices between the microparticles (5) free. The solution of resin,
hydrocolloid or monomer (7) accumulates at the points of contact of the microparticles.
The invention will now be further illustrated by the following examples.
Microfilters of example 1 - 4 are less resistant to organic solvents
but more easy to prepare. Microfilters of example 5 and 6 are especially useful
when labile macromolecules such as enzymes, antibodies or receptor proteins are
bound to microparticles. The solidification occurs under biocompatible conditions
with respect to temperature, acidity and tonicity. Microfilters of example 7 are
especially useful for covalent immobilization. Microfilters of example 8 are useful
when organic solvents are applied.
Production of biocompatible surface of the impermeable element and
production of microporous element by diffusion of nonsolvent.
A solution of 3% polyvinyl acetate (w/v) in acetone (molecular weight
500000, Cat. No. 38793-2; Aldrich, Steinheim G) was applied to the inner surface
of a 1ml-pipet tip (Cat. No. 00 30 001.311 Eppendorf, Hamburg G) and was drained
over blotting paper. After evaporating at ambient temperature and heating at 70░
C overnight, the tip was filled with 1 ml of 1 N-NaOH in water, and a partial reaction
was performed at 24░C for 30 min. Finally, the tip was washed and dried. In addition,
a liquid phase was prepared, by dissolving 1 g of poly (vinyl alcohol-co-ethylene)
of 44 mol % ethylene content (Cat. No. 41, 410-7; Aldrich, Steinheim G.) with 4
ml dimethyl sulfoxide at 100░ C. A volume of 3 Ál of liquid phase was taken up
at 40░ C. After closing the smaller aperture of the pipet tip, water was introduced
into the tip as a vapor. One hour later, 50 Ál of water was applied directly over
the solidifying liquid phase to obtain further diffusional precipitation. The flow
rate of the thus obtained filter element was 5,3 Ál/min (0,8 mm aperture; 500mbar).
Production by precipitation of resin with inclusion of microporous
100 Ág of reversed-phase material on silica gel (C18, 30
nm pore, 5Ám bead; Vydac, Hesperia, CA, USA) was sonicated with the polymer solution
(see example 1). This liquid phase was then treated further as described under
example 1. The flow rate was 1 ml/min (aperture 0,8 Ám; 1 bar). With silicon dioxide
(fumed silica, 14 nm size; Cat. No. S-5505, Sigma, Deisenhofen G.) as adsorbent,
instead of reversed-phase material, the flow-rate was 240 n/min.
Production by precipitation of semisynthetic polymer with inclusions
of a mixture of different types of microparticles.
A mixture was prepared from three different types of porous microparticles:
100 mg of anion exchanger based on silica gel (Adsorbex - SAX, Cat. No. 19845;
Merck, Darmstadt G.) 100 mg of cation exchanger (Adsorbex - SCX, Cat. No. 19846)
and 100 mg reversed-phase RP8 (Cat. No. 9362). After sonification with 1 ml of
dimethyl sulfoxide and centrifugation, the wet sediment was supplemented with 600
Ál of 15% cellulose propionate (average molecular weight 200 000; Cat. No. 18462-4;
Aldrich, Steinheim, G.) in dimethyl sulfoxide, prepared at 95░C. A modified Comfortip
(Cat. No. 30061; Eppendorf, Hamburg G) with biocompatible surface modification
was held pressed against the microporous-retainer-tool, filled with 35Ál of liquid
and layered with 80Ál of silicon oil and finally layered with 200Ál distilled water.
Positive pressure (at least 2 bar) was applied until the water phase passed finally
the microfilter. The flow rate was 10Ál/min. (1,5 mm aperture, 12 mm length; 7
Production by precipitation of semisynthetic polymer in the nanoliter
A volume of 200 nl of a 20% solution of cellulose propionate (mol.
weight 200 000) in dimethyl sulfoxide (w/v) was introduced into a gel loader tip
(Cat. No. 00 30 001.222; Eppendorf, Hamburg G). The aperture was closed temporarily
with silicon oil, and nonsolvent was introduced as a vapor from the other side.
The flow rate was 1,5 Ál/min (aperture 80 Ám; 100 mbar).
Production by crosslinking of reversible hydrogel in the presence
of channel-sparing nonsolvent at 0░C.
A solution of 5% (w/v) poly(vinyl alcohol) (molecular weight 124 000
- 186 000, 99% hydrolyzed; Cat. No. Aldrich, 36 306-5; Steinheim G.) in water was
prepared at 100░C. 4 ml of solution were mixed with 1 ml of 50 mM sodium tetraborate
4.7 and cooled to 0░C. Finally, 1g of enzyme-containing SAX particles (Adsorbex-SAX,
Merck, Darmstadt G.) were added. 40 Ál of this liquid-phase were filled into a
modified Comfortip with a biocompatible surface. The tip was pressed against a
microporous-retaining tool (see example 7) and was layered with 50 Ál silicon oil.
After applying positive pressure, migration of the silicon oil was achieved into
the granular bed. In order to achieve crosslinking, a final layer of 50 mM sodium
tetraborate pH 8.6 solution was forced through the Comfortip. The flow rate was
0,5 Ál/min (1,3 mm aperture; 7mbar).
Production by using a reversible hydrogel in the presence of a channel-sparing
One gram of DEAE-Si 300,10 Ám-size (Cat. No. 43536; Serva, Heidelberg
G) was mixed with 5 ml of a 2% agarose solution at 40░C. The agarose (low melt
preparative agarose; Cat. No. 162-0017; Bio-Rad Munich, G) had been dissolved at
90░C in 3% sucrose solution. Then, 100 Ál of liquid phase was taken up into a modified
standartip with biocompatible surface coating, as described before (Standartip
Cat. No. 30.003.004; Eppendorf, Hamburg G). The standartip was held pressed against
the microporous retaining tool, as described under example 7. After addition of
an upper layer of 200 Ál silicon oil at 40░C, positive pressure was applied until
150 Ál had passed into the microporous retaining tool. After removal of the tool,
solidification was achieved at 6░C. Finally the silicon oil was expelled by filtration
of 500 Ál sucrose solution. The filtration rate was 7 Ál/min (aperture 5 mm; 7
Production by polycondensation in presence of channel-sparing nonsolvent.
100 mg of silicagel (Lichrosorb Si 100, 7 Ám size; Cat. No. 9340;
Merck, Darmstadt, G.) were sonicated with 500 Ál of monomer solution (by vol: 5
parts of 1,4 - butanediol diglycidyl ether (Cat. No. 22,089-2; Aldrich, Steinheim
G.), one part dimethyl sulfoxide and one part ethylenediamine). 35 Ál of liquid
phase were taken up into a modified Comfortip (Cat. No. 30061; Eppendorf, Hamburg
G). The filled Comfortip was held pressed against a retaining tool consisting of
a microporous metal sieve (Cat. No. 12550812; Bischoff, Leonberg) located on a
3 mm thick filter paper (Cat. No. 2727; Schleicher and SchŘll, Dassel, G). After
layering 80 Ál of silicon oil onto the liquid-phase, positive pressure was applied
(at least one bar) until 30 Ál of silicon oil were flown through the Comfortip.
After removal from the retaining tool, solidification was achieved by curing in
a closed system. Finally, the micro filter was washed with distilled water and
suitable buffer. The flow rate was 32 Ál/min (1,3 mm aperture; 1 bar).
When using nonporous silicon dioxide (1-5 Ám, Cat. No. S-56631; Sigma,
Deisenhofen G.) instead of silica gel as adsorbent, the flow rate was 1 Ál/min
(aperture 1,5 mm; volume 10 Ál, Comfortip 1 bar).
Production by polymerisation in the presence of channel-sparing nonsolvent.
100 mg of silica gel type anion exchanger of HPLC quality (DEAE-Si
300, 10 Ám; Cat. No. 43536; Serva, Heidelberg G) were sonicated with ice-cold 500
Ál monomer solution (By weight: 26% acrylamide; 0,7% bis acrylamide; 170 mM Tris
HCl pH 8,8; 0,05% tetramethyl ethylenediamine (Cat. No. 8133; Sigma, Deisenhofen)
0,08% sodium persulfate).
All further steps were done as described in example 7 except that
the temperature was held between 0-2░C as during the flushing step with silicon
oil. Finally, solidification was achieved by curing at 45░C. The flow rate was
20 Ál/min (aperture 1 mm; 600 mbar).
Production using hydrogel and microparticles with reformation of channels
by means of drying.
One ml of a 2% (w/v) solution of agarose (Bio-Rad, Munich G.) was
mixed and sonicated with 200 mg of microporous titan dioxide (YMC-Gel, TIAOS 20
NP; YMC Europe, Schermbeck, G.) at 40░C. At the same temperature, 50 Ál of liquid
was taken up into a pipet tip that was provided with biocompatible surface coating
(see example 1) and drained over a microporous-retaining-tool (see example 7).
The pressure was adjusted in a way that flow was stopped when the microparticles
were just beginning to be exposed. Solidification was achieved at 6░C. Drying was
done at ambient temperature and finally over P2O5, at 1 mb.
The flow rate was 33 Ál/min (1 bar).
Isolation of multimolecular complexes consisting of oligonucleotides
and specific macromolecules, noncovalently bound together.
A labelled oligonucleotide probe was incubated with nuclear extract,
and 8 Ál were filtered over a polyacrylamide - DEAE microfilter (example 8) which
was washed with 40 Ál buffer. The free oligonucleotide was retained and the complexed
form was obtained in the eluate within some minutes.