Patent Description:
Rapid diagnostic testing at the site of a patient, so-called 'point-of-care' (POC) testing, is essential to provide healthcare when a fully equipped laboratory is not accessible. In developing countries, suitable POC diagnostics could yearly save millions of lives by early diagnosis of a small number of treatable conditions identified by the World Health Organization (WHO). As a result of the inverse correlation between the number of lives saved and the level of diagnostic infrastructure required, the WHO defined guidelines for viable developing world diagnostics that underscore the need for low-cost, disposable assays that require minimal user-dependent steps and are equipment-free (i.e. without the need for external readers, electricity, electronic timers, etc.).

Lateral flow tests (e.g. home pregnancy tests) are one of the few technologies that meet these criteria. In these tests, the liquid sample wicks through a porous paper-like membrane driven by capillarity and readout occurs by eye (e.g. the appearance of colored lines). Notwithstanding their success, lateral flow tests are typically not quantitative and their sensitivity is limited as chemical signal amplification is not possible. More sensitive amplification-based analyses (e.g. enzyme-linked immunosorbent assays, ELISAs) require a precisely timed sequence of steps: pretreatment of the sample (e.g. plasma separation), addition of reagents, washing steps, incubation and result readout. Sparked by the success of lateral flow strips, there have been many attempts to automate a timed assay sequence in so-called 'passive microfluidics' that are driven solely by capillary flow. These approaches are based on defining 3D flow paths within stacks of porous membranes that are individually functionalized with hydrophobic/hydrophilic patterns and assay reagents [<NPL>; <NPL>;<NPL>;<NPL>; <NPL>;<NPL>; <NPL>]. While these passive microfluidics are undoubtedly low-cost, integrating amplification-based assays requires bonding many compressible porous layers without affecting their fluid transport properties [<NPL>]. The resulting interlayer contact issues lead to unpredictable or defective devices (~<NUM> % for a <NUM>-layer assembly) [<NPL>].

Currently there is still no ideal technology platform for diagnostic testing in developing countries and there is a need for a method for providing a novel POC. The ideal POC test would combine the best of both lateral flow and ELISA tests. In addition, given the proportions of the targeted challenge, scalable fabrication is essential.

Since the performance issues in the passive multistep assays tested thus far seem intrinsic to their manufacturing method (i.e. bonding stacks of compressible porous layers without altering fluid transport properties), the laminated membrane format is abandoned. Instead, a radically new method is proposed to fabricate "monolithic" passive microfluidics, namely through 3D printing (3DP). Thus far, 3DP has mainly been employed in applications where geometrical shape defines the printed object function (e.g. product design). Other applications for 3DP include to produce products enabling chemical or biological reactions (<CIT>, <CIT>) or for drug delivery applications (<CIT>, <CIT>).

Inkjet 3D printing of microfluidics has been performed by <NUM>-photon writing methods wherein a pattern of channels is carved out by a pulsed laser source. <CIT> describes a method wherein a microfluidic device is made using a hydrophobic material such as PTHPMA for the bulk of the device and a hydrophobic material with a hydrolysable group and a photoacid generator for the sections defining the channels. After 3D printing the device is exposed to UV which renders the material with the hydrolysable group hydrophilic.

<CIT> discloses layered objects wherein water based suspensions of fine powders are deposited. Channels are made by selectively adding a hydrophobic material. This material repels a subsequently added water based suspensions, leaving a gap. The gap is filled with a secondary sacrificial material. After completing of the object, the object is sintered to solidify the powder and to remove the secondary material.

The present invention provides a novel method for providing a porous subject suitable for 'passive microfluidics', i.e. driven by capillary flow.

The methods of the present invention have the advantage that the devices can be prepared without the need of laser equipment.

The methods of the present invention have the advantage that the devices can be prepared without the need of a UV source, or with material that is not compatible with UV radiation.

The methods of the present invention have the advantage that the devices can be prepared using one type of particulate material, which does not require reactive groups on a particulate material.

The methods and devices of the present invention have several differences and advantages over prior at methods such as disclosed in <CIT>.

In the methods of the present invention one type of particular material is use, making the use of a second material sacrificial unnecessary. The channels in a device obtained by the methods of the present are not empty channels, but still contain particles. Hydrophilic liquids flow via the cavities in between the particles, leading to a more intimate between fluid and particle wall. This has the advantage that the interaction between an analyte and capturing/detecting agent will be more efficient than in channels where the interaction only occurs at the wall of the channel.

The methods of the present invention do not require heat treatments to remove the secondary material. This allows as well to use particulate material which is heat sensitive as to include in the channels of the device heat labile compounds. Methods wherein sintering is used to solidify the powder and/or to remove secondary material restrict the choice of materials and fields of applications.

This results in at the one hand particles bound by binder surrounded by hydrophobic material and at the other hand particle bound by binder surrounded by a hydrophilic environment. In step <NUM>, the bed is lowered to receive a further layer of powder.

As used herein, "three-dimensional (3D) printing" refers to a process that sequentially stacks layers each having a predetermined cross-sectional shape to produce 3D objects layer-by-layer from digital designs. The basic process of 3D printing has been described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>. The patents listed above disclose a detailed description of 3D printing.

There are different 3D printing technologies; their main differences concern the way in which the various layers are printed. Some methods, in order to produce the various layers, use materials that melt or soften. Some examples of such technology are "Selective Laser Sintering" (SLS) or "Direct Metal Laser Sintering" (DMLS) or "Fused Deposition Modeling" (FDM). Within the context of the present invention, a preferred 3D printing method is an inkjet 3D printing system. The printer creates the model one layer at a time by spreading a layer of powder (e.g. plaster or resins) and printing a binder in the cross-section of the part using an inkjet-like process. This is repeated until every layer has been printed. This technology allows the printing of full color prototypes, overhangs, and elastomer parts. The strength of bonded powder prints can be enhanced with wax or thermoset polymer impregnation.

The materials usually used for 3D printing are: plastic materials, for example thermoplastic polymers (for example for SLS and FDM), metals, sand, glass (for example for SLS), photopolymers (for example for stereolithography), laminated sheets (often of the paper type) and relative glues, titanium alloys (for example for "Electron beam melting" or EBM), resins, clays, ceramic, etc..

"Porous" or "porosity" refers to the void spaces in a material. Porosity can be expressed as the fraction of the volume of voids over the total volume of the 3D printed material. Parameters which influence the porosity of the 3D printed material are for example the size of the particulate matter, the shape of the particulate matter, the size distribution of the particulate matter, the preparation of the powder bed, the type and amount of binder and the conditions of connecting the particulate matter with a binder. In contrast to e.g. an extruded material wherein the porosity is caused by air bubbles in a material, the 3D printed materials of the present invention are typically aggregates of globular particular material, whereby the contacts between individual particles are melted together in a less or greater degree, depending on the binding conditions. The porosity is thus generated by interconnected cavities inbetween the individual particular which form a network throughout the 3D printed material. The fact that a material has a certain porosity whereby cavities make a network through the 3D printed material does not mean "per se" that the material allows gas or liquid transport. As illustrated in the present invention, the surface of the particulate matter prevails over the porosity for determining whether a liquid will be able to migrate through the porous material.

"Capillary transport" (or capillarity, capillary motion, or wicking) as used herein refers to the ability of a fluid such as a liquid to flow in narrow spaces without the assistance of, or even in opposition to, external forces like gravity. It occurs because of intermolecular forces between the liquid and surrounding solid surfaces. If the diameter of the channels in a porous network are sufficiently small, then the combination of surface tension (which is caused by cohesion within the liquid) and adhesive forces between the liquid and the channel walls act to propel the liquid.

"Hydrophobic" as used herein refers to repelling water, such as a hydrophobic surface wherein said surface repels water. The hydrophobicity of a surface can be measured, for example, by determining the contact angle of a drop of water on the surface. On an extremely hydrophilic surface, a water droplet will completely spread over the surface and exhibit a contact angle of approximately <NUM>°. This situation arises for surfaces that absorb water or have a high affinity for water. A hydrophilic surface as used herein refers to a surface which can have a water contact angle of less than about <NUM>°, more preferably less than about <NUM>°, even more preferably less than about <NUM>°. For example, many hydrophilic surfaces have contact angles from around <NUM>° to <NUM>°. A hydrophobic surface as used herein thus refers to a surface that has a water contact angle greater than about <NUM>°, preferably greater than about <NUM>°, more preferably greater than about <NUM>° or above. A superhydrophobic surface is for example considered to exhibit a contact angle greater than about <NUM>°, and a non-wetting surface has a contact angle of <NUM>°. The contact angle can be a static contact angle or dynamic contact angle. A dynamic contact angle measurement can include determining an advancing contact angle or a receding contact angle, or both. A hydrophobic surface having a small difference between advancing and receding contact angles (i.e., low contact angle hysteresis) can be desirable. Water can travel across a surface having low contact angle hysteresis more readily than across a surface having a high contact angle hysteresis. A measurement method for powder contact angles is described in <NPL>, wherein a porous media column is placed in contact with water, allowing the fluid to wick through. The column weight change is recorded during the wicking process until water front stops. The contact angle is determined from the mass change as a function of time.

Flat surfaces for contact angle determination can be generated by spincoating. PMMA surfaces can be obtained by spincoating PMMA solutions in acetone onto glass surfaces. For contact angle measurements, a deionized water drop is deposited onto the spincoated PMMA surface. Immediately after drop deposition, a picture can be taken with CAM <NUM> setup (KSV, NIMA, FL) to measure the contact angle using CAM <NUM> associated software [<NPL>].

The term "powder or powdered material" as used herein refers to polymer powder and can be a single compound or a mix of different polymers and includes, but is not limited to, the group of PMMA, polystyrenes and their copolymers or block copolymers. Powder grain size is typically in the <NUM>-<NUM> range. The grain size can be mono- or polydisperse. The grain shape can be spherical, regular or irregular. As can be appreciated, the size and shape have an effect on the porosity of the material.

In the methods and device of the present invention it is the aim to use a hydrophilic particular materials, which requires to treatment in the regions forming the chambers and channels, whereas the particles forming the bulk of the device are treated with a hydrophobizing agent.

The embodiment wherein hydrophobic powder material is used which is rendered hydrophilic in the regions forming the channels and chamber is possible but less preferred.

"Depositing outlet" as used herein refers to a printhead nozzle or outlet or other deposition mechanism outlet. In the context of the present invention, the term depositing outlet typically refers to a deposition mechanism for depositing a printing liquid on a powder bed.

"Binder", or "binding agent" as used herein is an agent suitable for binding the powder material and is comprised in the printing liquid. Typically, a binder is at least one organic solvent which is capable of at least partially dissolving the powder and which includes, but is not limited to, toluene, chlorinated solvents (such as for example but not limited to chloroform or dichlorobenze), ethyl lactate, ethyl acetate, dioxane, acetophenone, dichlorobenzene, dimethylformamide (DMF), tetrahydrofurane (THF), acetonitrile, hexane, ethanol, hexanol, hexyl acetate, or ketones (such as for example but not limited to acetone, acetophenone, butanone or pentanone), esters (such as for example but not limited to ethyl acetate, ethyl lactate, isopropyl acetate or hexyl acetate), dioxane, acetonitrile, or any combinations or mixtures thereof. A selection of different binders/binder classes has also been described in [<NPL>].

The "printing liquid" as used herein refers to any liquid that is destined to be deposited on the powder bed, preferably via a depositing outlet. Printing liquids may comprise water, an organic or anorganic solvent, a binder, a reactant, a hydrophobizing agent, a hygroscopic compound or any combination of the previous. When hydrophobizing agents are added to the printing liquid, the printing liquid is referred to as hydrophobizing printing liquid.

"Reactants" as used herein refers, within the context of the present invention, to compounds for capturing and/or detecting analytes contained in the hydrophilic fluid sample applied to the printed microfluidic network. Reactants are preferably introduced in the microfluidic network by selectively depositing a printing liquid comprising the reactant at predetermined positions on the powder bed. Reactants include, but are not limited to, pH indicators, colored indicators, nucleic acid sequences or proteins like antibodies and enzymes.

"Hydrophobizing agents" as used herein refers, within the context of the present invention, to compounds which prevent a hydrophilic fluid to wick through predetermined sections in the microfluidic network to which the hydrophobizing agent was introduced, preferably by selectively depositing a printing liquid comprising the hydrophobizing agent on the powder bed, thus creating hydrophobic sections or walls surrounding sections enabling the capillary transport of hydrophilic fluids. Hydrophobizing agents as used herein include, but are not limited to, waxes, silanes, alkyl and alkenyl ketene dimers, acid anhydrides, including alkyl anhydrides and alkenyl succinic anhydride, hydrophobic polymers, hydrophobic particles, fluorinated molecules, or molecules containing apolar hydrocarbon moieties, or any combinations thereof. A "hygroscopic compound" is a compound that readily absorbs water molecules from its environment by either absorption or adsorption and as used herein refers, within the context of the present invention, to compounds which increase the hydrophilicity of predetermined sections in the microfluidic network to which the hygroscopic agent was introduced, preferably by selectively depositing a printing liquid comprising the hygroscopic agent on the powder bed, thus increasing the flow speed of the capillary transport of the hydrophilic fluid in said predetermined sections. Hygroscopic compounds as used herein include, but are not limited to, ionic salts, sugars or polyethyleneglygols (PEGs).

Additionally, the surface tension and viscosity of the printing liquid mixture may be altered to match the printhead surface tension and viscosity requirements (<NUM>,<NUM> - <NUM>,<NUM> N/m or <NUM> - <NUM> dyn/cm and <NUM>,<NUM> - <NUM>,<NUM> Pa s or <NUM> - <NUM> cP, respectively) by adding surfactants like fatty alcohol ethoxylates and alkylphenol ethoxylates and high viscosity compounds such as polyethylene glycols, glycerol, and high alkanes, respectively.

In a first object the present invention presents a method according to claim <NUM> for three dimensional printing of a porous object, said object comprising hydrophobic sections delineating one or more sections enabling the capillary transport of hydrophilic fluids.

In a preferred embodiment of the present invention, said method involves the use of a print device comprising a powder bed plate, means for spreading a powder material on said powder bed plate in order to provide a powder bed within a defined area, one or more depositing outlets for depositing a printing liquid on said powder bed and means for repositioning said depositing outlets with respect to the surface of said powder bed, wherein said printing device allows for selectively depositing one or more printing liquids with varying compositions and physicochemical properties at predetermined positions on the powder bed; said method comprising the steps of: (a) providing one or more printing liquids wherein at least one printing liquid comprises a hydrophobizing agent, referred to as hydrophobizing printing liquid and at least one printing liquid comprises an agent suitable for binding the powder material of step (b), referred to as binder; (b) spreading a layer of a wettable powder material over said powder plate in order to create a powder bed; (c) depositing one or more of said printing liquids at predetermined positions on said powder bed, wherein the depositing of said printing liquid comprising a binder results in the binding of the powder particles at its depositing position and wherein the selective depositing of said hydrophobizing printing liquid on the powder bed provides for said hydrophobic sections of said porous object.

Typically, said selective depositing is controlled by a computing unit wherein said computing unit controls the movement of the depositing outlets relative to the powder bed and the timing and position of the deposition of a printing liquid on said powder bed from said depositing outlets, for example using computer readable data indicating said predetermined positions for selective deposition. Typically, said powder bed is a substantially horizontal powder bed which is largely, but not necessarily wholly horizontal. Substantially horizontal as used herein refers to horizontal to less than <NUM>° from horizontal, more preferably from horizontal to less than <NUM>° from horizontal. Said powder bed may however also be a powder bed with an oblique angle of more than <NUM>° from horizontal.

In another preferred embodiment of the present invention, each of said one or more printing liquids has a different composition and said selective depositing of said one or more printing liquids at predetermined positions on the powder bed provides specific physical and/or chemical properties to different portions of said object.

The powder bed surface could also be altered by powder suction for creating cavities such as described in <NPL>.

In another preferred embodiment of the present invention, said hydrophobizing printing liquid comprises a hydrophobizing agent selected from the group comprising waxes, silanes, alkyl and alkenyl ketene dimers, acid anhydrides (such as but not limited to alkyl anhydrides and alkenyl succinic anhydride), hydrophobic polymers, hydrophobic particles, fluorinated molecules, molecules containing apolar hydrocarbon moieties and any combination thereof. Preferably, said hydrophobizing printing liquid comprises a hydrophobizing agent selected from the group consisting of waxes, silanes, alkyl and alkenyl ketene dimers, acid anhydrides (including but not limited to alkyl anhydrides and alkenyl succinic anhydride), hydrophobic polymers, hydrophobic particles, fluorinated molecules, molecules containing apolar hydrocarbon moieties and any combination thereof. More preferably, said hydrophobizing printing liquid comprises a hydrophobizing agent selected from the group consisting of alkenyl succinic anhydrides, alkyl and alkenyl ketene dimers and waxes and any combinations thereof.

It is further preferred that said hydrophobizing printing liquid further comprises a binder. Typically, said binder is selected from the group comprising water, ketones (such as but not limited to acetophenone, hexanone, propanone, butanone, methylethylketone or pentanone), benzene, xylene, toluene, chlorinated solvents (such as but not limited to chloroform or dichlorobenzene), esters (such as but not limited to ethyl acetate, ethyl lactate, isopropyl acetate or hexyl acetate), dioxane, acetonitrile, dimethylformamide (DMF), ethylene dichloride, tetrahydrofurane (THF), alcohols (such as but not limited to ethanol, hexanol or phenol), cresols, fluorinated solvents, dimethylacetamide, lithium chloride, N-methylporpholine N-oxide, glycol ether, DMSO, salts or any combinations thereof. Preferably, said binder is selected from the group consisting of acetophenone, butanone, hexanone, propanone, methylethylketone, pentanone, toluene, chloroform, ethyl acetate and any combinations thereof. More preferably, said binder is selected from the group consisting of acetophenone, butanone, hexanone, propanone and any combinations thereof.

Typically, said print device comprises at least one outlet for depositing said at least one printing liquid on said powder bed and wherein each of the at least one respective printing liquids is deposited on the powder bed from a different depositing outlet. Preferably, said print device comprises at least one outlet for depositing printing liquids, for example at least one, <NUM>, <NUM>, <NUM>, <NUM> or more outlets for depositing printing liquids.

The powder material used in the method of the present invention typically comprises an organic and/or an inorganic particulate material having a particle size varying between <NUM> and <NUM>. More preferably, said particulate material has a particle size of at least <NUM>, for instance at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or at least <NUM>. It is further preferred that said particulate material has a particle size of not more than <NUM>, for instance not more than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or not more than <NUM>. Even more preferably, said particulate material has a particle size of about <NUM> to <NUM>, such as of about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

In the methods of the present invention the particulate matter is applied as a dry powder, not as a suspension. After deposition of the powder liquids are added on the deposited powder such as the binding liquid, hydrophobizing agents, and reagents (such as enzymes, antibodies), as described in more detail in the present applications. Reagents such as buffers and proteins may deposited allowing transport with the hydrophilic fluid upon use. Alternatively, reagents such as antibodies are immobilized to allow a sandwich assay, or chromophoric enzymatic substrates are added to determine enzyme activity.

In another preferred embodiment of the present invention, said powder material comprises a polymer and said binder is a solvent for said polymer (Table <NUM>). Typically, said polymer is selected from the group comprising polymethyl methylacrylate (PMMA), acrylonitrile butadiene styrene (ABS), poly lactic acid (PLA), poly styrene (PS), poly vinyl alcohol (PVA), nylon, cellulose, nitrocellulose, cellophane, and their copolymers or block copolymers. Preferably, said polymer is selected from the group consisting of PMMA and PS and their copolymers or block copolymers.

Typically, said binder is selected from the group comprising water, ketones (such as but not limited to acetophenone, hexanone, propanone, butanone, methylethylketone or pentanone), benzene, xylene, toluene, chlorinated solvents (such as but not limited to chloroform or dichlorobenzene), esters (such as but not limited to ethyl acetate, ethyl lactate, isopropyl acetate or hexyl acetate), dioxane, acetonitrile, dimethylformamide (DMF), ethylene dichloride, tetrahydrofurane (THF), alcohols (such as but not limited to ethanol, hexanol or phenol), cresols, fluorinated solvents, dimethylacetamide, lithium chloride, N-methylporpholine N-oxide, glycol ether, DMSO, salts or any combinations thereof. Preferably, said binder is selected from the group consisting of acetophenone, butanone, hexanone, propanone, methylethylketone, pentanone, toluene, chloroform, ethyl acetate and any combinations thereof. More preferably, said binder is selected from the group consisting of acetophenone, butanone, hexanone, propanone and any combinations thereof.

Preferably, said powder material comprises a polymer, wherein said polymer is polymethyl methacrylate (PMMA) and wherein said binder is selected from the group consisting of acetophenone, butanone, propanone, hexanone and any combinations or mixtures thereof.

Said binding agent may alternatively also polymerize the powder material or initiate a polymerization to form bonds.

In another preferred embodiment of the present invention, said method of the present invention involves the use of said print device, wherein said print device is a 3D printing device comprising means for lowering said powder bed plate and wherein said method comprises in addition to steps (a), (b) and (c) of the first embodiment of the present invention, the additional steps of (d) lowering the powder bed; and (e) repeating steps (c) and (d) until the object is formed, whereby at each repetition of step (c) an additional layer of the porous object is formed and wherein at least part of the powder material of the powder bed is bound to bound material of the layer formed in the previous step (c) wherein said binding of layers results from the directed depositing of said printing liquid comprising said binder, and wherein said object comprises at least one section that is accessible for applying a hydrophilic fluid sample and that enables capillary transport of hydrophilic fluids.

Typically, said section for applying a hydrophilic fluid sample connects with an internal section of said object enabling capillary transport of hydrophilic fluids.

The method according to the present invention may be used for printing a porous object wherein said sections of said porous object enabling capillary transport of hydrophilic fluids are interconnected in a microfluidic network suitable for the capillary transport along a predefined path of a hydrophilic fluid sample applied on said at least one accessible dedicated section of said object, said dedicated section being part of said microfluidic network.

In the devices of the present invention, the hydrophilic particulate material remains present in the regions forming the channels and/or chamber for capillary transport. The powder material is present herein as stacked balls with cavities inbetween them. When binding agent is used, the particulate material form a cluster of interconnected particles with inbetween them a network of cavities, allowing a capillary flow.

Compared to prior art devices wherein "empty" channels are formed, the presence of particulate matter within the channel generates more interaction between fluid and particles. Accordingly when the particles are coated with a reagent, the interaction between an analyte in a sample and a reagent on a particle will be more efficient that in a channel wherein only the surface of the cylindrical channel is coated with a reagent.

The presence of the particles within a channel also allows the manipulation of fluids beyond the mere capillary action of the fluid. For example the particles in a region of a channel can be pretreated with e.g. a surfactant such as Tween™ acting as a plug, which temporarily delays the flow of the fluid. This allows for example that an chemical reaction can be performed at a certain place for a predetermined period. This concept has been already published by <NPL> and can be introduced in this type of devices which are prepared without sintering, or high temperature treatment.

Said at least one dedicated section of said object for applying a hydrophilic fluid sample may consist of unbound material, partially bound material or unbound material.

It is preferred that the external surfaces which do not contain said at least one dedicated section for applying a hydrophilic fluid sample of said printed porous 3D object consist of bound material. Such bound external surfaces can be obtained when during the printing of said object a printing liquid comprising a binder is deposited on the powder surfaces that are destined to become part of said external surface of said 3D object. The internal part of such object, i.e. the part of the object enclosed by said bound external surfaces, or said at least one dedicated section for applying a hydrophilic fluid sample, may comprise sections containing unbound or partially bound powder material.

Preferably, said microfluidic network comprises microfluidic chambers and/or channels. More preferably, said microfluidic chambers and/or channels have a height of about <NUM> to about <NUM>, for example of at least <NUM> and not more than <NUM>, e.g. of <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>. Even more preferably, said microfluidic chambers and/or channels have a height of about <NUM> to about <NUM>.

Preferably, said microfluidic chambers and/or channels have a width of about <NUM> to about <NUM>, for example of at least <NUM> and not more than <NUM>, e.g. of <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>. Even more preferably, said microfluidic chambers and/or channels have a width of about <NUM> to about <NUM>.

Preferably, said microfluidic network comprises zones comprising compounds for capturing and/or detecting analytes contained in said hydrophilic fluid sample applied on said at least one dedicated section, wherein said compounds for analyte detection and/or capture are introduced in said network by selectively depositing a printing liquid comprising said compound for analyte detection and/or capture at predetermined positions on said powder bed.

Said compounds may be immobilized by a covalent bond of said compound to the printed powder. Said compounds may also not be covalently bound to the printed powder which enables the compound to mobilize through the microfluidic network in the hydrophilic fluid.

Said analyte capturing or detection compound may be for example, but is not limited to, a pH indicator, an antibody (such as but not limited to a labeled antibody or tagged antibody), a DNA molecule, a RNA molecule, an enzyme, an enzyme substrate, a color indicator, an enzyme cofactor, an enzyme inhibitor, an antibody-enzyme conjugate, a chemical reactant, or a buffer.

Preferably, said analyte detection compound is selected from the group consisting of a pH indicator, a color indicator, an antibody, DNA molecule, a RNA molecule, an enzyme, an enzyme substrate, a color indicator, an enzyme cofactor, an enzyme inhibitor, an antibody-enzyme conjugate, a labeled antibody, a chemical reactant and a buffer.

Said microfluidic network may also comprise zones for controlling the flow rate of the hydrophilic liquid in said network.

Said zones may for example be off-channel reservoirs or channels that need to be filled before the hydrophilic fluid sample continues in the microfluidic network channel direction, because of which the flow of the hydrophilic fluid in the channel network is delayed.

Said zones may also be partially hydrophobic zones created by depositing a partially hydrophobizing printing liquid comprising a binder resulting in the binding of the powder particles at said zone and wherein the selective depositing of the partially hydrophobizing printing liquid on the powder bed provides for said partially hydrophobic zones of said porous object, and wherein said partially hydrophobic zones control or delay the flow of the hydrophilic fluid sample in said network.

Said zones may also comprise compounds or elements that act as triggers or timers to control the flow or the flow rate of the hydrophilic fluid through said network, wherein said compounds for flow or flow rate control are introduced in said network by selectively depositing a printing liquid comprising said compound for flow or flow rate control at predetermined positions on said powder bed. Said compound may for example be a hydrophobic compound, such as hydrophobic boronic esters that are printed into a defined hydrophilic section of the network creating a blocking section which blocks the advancement of the hydrophilic fluid through the network, and wherein said hydrophobic compound decomposes into hydrophilic fragments upon contact with a decomposing molecule, such as H2O2, therefore degrading the blocking section and allowing the hydrophilic fluid to further advance through the network [<NPL>;<NPL>].

Said zones may also comprise amended pore size or porosity for example created by depositing a printing liquid comprising a binder with a different binding rate, wherein said zones with modified pore size or porosity control, fasten or delay the flow of the hydrophilic fluid sample in said microfluidic network.

In a preferred embodiment of the present invention, said porous object is a device for liquid handling.

In another preferred embodiment of the present invention, said porous object is a point of care diagnostic device wherein said device comprises a read-out section that is part of said microfluidic network and that is adapted to provide a signal depending on either the presence, absence or concentration of an analyte in a hydrophilic fluid sample applied on said dedicated section.

In a second object, the present invention presents a porous object comprising hydrophobic sections delineating one or more sections enabling the capillary transport of hydrophilic fluids prepared by three-dimensional printing according to the method of any one of the embodiments of the first object of the present invention.

In a preferred embodiment of the present invention, said object is a liquid handling device, according to claim <NUM>.

In another preferred embodiment of the present invention, said object is a point of care diagnostic device.

The combination of a roller and a scraper blade allows powder spreading onto the printing bed. The roller rotates in the direction opposite to the spreading direction. A printout support layer composed of loose powder is created by spreading about <NUM> to about <NUM> homogeneous and dry powder layers of about <NUM> - <NUM> thickness. In between each layer deposition, the printing bed is lowered by one layer thickness. However, thicker supports are also possible.

At the start of the printing process, another homogeneous and dry powder layer is deposited onto the printout support layer ("powder spreading" or step <NUM>, <FIG>). The roller rotation direction remains opposite to the spreading direction. Layer thickness is about <NUM> - <NUM>.

Inkjet printheads allow printing liquid deposition onto the previously deposited layer. The jetted drop volume is about <NUM> to about <NUM> pL. Printing liquid is deposited on the powder layer within an area determined by a digital computer model of the object to be printed. Printing liquids comprising a binder combined with or in absence of an hydrophobizing agent are respectively deposited onto predetermined positions or areas of the powder bed, matching object sections labelled in said computer model as "hydrophobic area" and "hydrophilic area", respectively ("binder printing" or step <NUM>, <FIG>). Alternatively, in step <NUM>, a printing liquid comprising a binder combined with a hydrophobizing agent is deposited onto a predetermined "hydrophobic area" of the powder bed, whereas no printing liquid is deposited onto a predetermined "loose powder" area of the powder bed (<FIG>). Said "loose powder" area also allows capillary transport of hydrophilic fluids through said "loose powder" channel with hydrophobic walls. Alternatively, in step <NUM>, a printing liquid comprising a binder combined with a hydrophobizing agent, or a printing liquid in the absence of a binder but combined with a hydrophobizing agent, may be deposited onto a predetermined "hydrophobic area" of the powder bed; and/or a printing liquid comprising a binder in the absence of a hydrophobizing agent, or a printing liquid in the absence of a binder and a hydrophobizing agent, may be deposited onto a predetermined "hydrophilic area"; and/or no printing liquid may be deposited onto a predetermined "loose powder" area of the powder bed.

The inkjetted printing liquid comprising a binder is allowed to spread in between the powder layer grains via capillarity forces. The polymer grain surfaces are partially softened and dissolved by binder. The result is a porous network of connected particles in the area where binder was deposited. Hydrophobizing agent present in the hydrophobizing printing liquid remains onto the powder grain surface, resulting in a porous network with internal hydrophobic surfaces ("printed layer" or step <NUM>, <FIG>).

The printing bed is then lowered by one layer thickness, between about <NUM> and <NUM> ("bed lowering" or step <NUM>, <FIG>).

Previously described steps <NUM>, <NUM>, <NUM> and <NUM> (<FIG>) are repeated until all predefined object sections from the computer model have been translated into a corresponding printed powder layer. During this process, printing liquid inkjetted onto the top layer spreads to previous layers, resulting in the bonding of these layers and eventually a porous 3D object with precise control over the "hydrophilic" and/or "hydrophobic" and/or "loose powder" functionalization of both internal and external sections of said 3D object (<FIG>, B and C).

After printing completion, the powder bed can be heated to improve solvent residue evaporation. This step is usually performed at about <NUM> for allowing quick solvent evaporation. However, any temperature between about <NUM> to <NUM> is suitable for solvent evaporation, depending on the composition of the printing liquid components (e.g. temperatures between about <NUM> to <NUM> are used when using printing liquids comprising sensitive reactants, such as antibodies). After printing, printouts can be placed at <NUM> for 1hr for allowing the hydrophobizing agent to quickly cure (i.e. be activated). However, printouts can also be incubated at room temperature for <NUM> to <NUM> days to allow curing at room temperature.

The methods of the present invention thus allow to prepare devices for liquid handling without sintering. The main reason for a drying step is to evaporate solvants used as binder agent or used as hydrophobizing agent. The use of temperatures below <NUM>, below <NUM>, or at room temperature between <NUM> and <NUM>) allows the use of heat sensitive particulate material as well as heat sensitive reagents such as proteins.

When in step <NUM> of EXAMPLE <NUM>, a printing liquid comprising a binder combined with a hydrophobizing agent is deposited onto a predetermined "hydrophobic area" of the powder bed, and a printing liquid comprising a binder in the absence of a hydrophobizing agent is deposited onto a predetermined "hydrophilic area", and no printing liquid is deposited onto a predetermined "loose powder" area of the powder bed, a porous object is printing containing a microfluidic network comprising channels, allowing capillary transport of hydrophilic fluids, which are either containing loose powder (unsintered channels), or can be bound (hydrophilic areas), and wherein the unsintered channels are connected/adjacent to the hydrophilic areas (hydrophilic caps) (<FIG>).

The procedure remains identical as described in EXAMPLE <NUM>, with the addition of an extra functional printing liquid comprising an additional functional component. When in step <NUM> of EXAMPLE <NUM>, a printing liquid comprising a binder in absence of a hydrophobic agent is deposited onto a predetermined "hydrophilic area", and a printing liquid comprising a hydrophobic agent and a binder is deposited onto a predetermined "hydrophobic area" and a printing liquid comprising a binder and an hygroscopic component in absence of a hydrophobic agent is deposited onto a predetermined "hygroscopic area", a porous object is printed containing a microfluidic network comprising channels allowing capillary transport of hydrophilic fluids which are bound (hydrophilic areas) and may contain a hygroscopic component which can increase the flow speed of the capillary transport of the hydrophilic fluid in said predetermined hygroscopic sections (<FIG>).

The procedure remains identical as described in EXAMPLE <NUM>, with the addition of an extra functional printing liquid comprising an additional functional component.

When in step <NUM> of EXAMPLE <NUM>, a printing liquid comprising a binder in absence of a hydrophobic agent is deposited onto a predetermined "hydrophilic area", and a printing liquid comprising a hydrophobic agent and a binder is deposited onto a predetermined "hydrophobic area", and a printing liquid whether in the presence or absence of a binder but in the absence of a hydrophobic agent, comprises an additional reactive component is deposited onto a predetermined "reactive area", a porous object is printed containing a microfluidic network comprising channels allowing capillary transport of hydrophilic fluids which are either bound (hydrophilic areas) and/or may contain a reactive component which allows for capturing and/or detecting analytes contained in the hydrophilic fluid sample in said predetermined reactive sections (<FIG>).

Alternatively, said reactive area may be printed by "in situ mixing", in which a printing liquid comprising the reactive component in the absence of a binder or a hydrophobic agent is printed onto the predetermined reactive area, and a printing liquid comprising a binder in the absence of a hydrophobic agent is printed on the same predetermined area, but in which both printing liquids are printed from a different print head.

3D printing device: For the purpose of this example a Projet ™ <NUM> pro (3D Systems, Rock Hill, USA) 3D printing was used, which comprises multiple exchangeable printing heads, wherein each printing head corresponds to a printing liquid reservoir operationally connected to an outlet channel for depositing this printing liquid on the powder bed.

Powder: The powder material used in the printing of the 3D object according to this example was Poly(methyl methacrylate) (PMMA) powder (grain size: <NUM>-<NUM>).

Printing liquids: Following mixtures of organic solvents were found suitable for PMMA: acetophenone/acetone, acetophenone/butanone and acetophenone/propanone. By appropriately mixing these solvent combinations, printing liquids with binder properties were obtained having suitable viscosity and surface tension for use in the printing heads of the Projet™ <NUM> pro 3D printing device. For example, mixing acetophenone and acetone between a <NUM>/<NUM> to a <NUM>/<NUM> ratio provided a suitable printing liquid for binding the PMMA powder without conferring hydrophobic properties to the bound powder section ("hydrophilic binder"). A printing liquid with both binding and hydrophobizing properties was provided by adding between <NUM> to <NUM> of N-Octadecylsuccinic acid anhydride (hydrophobizing agent) per <NUM> of any of the preceding organic solvent mixtures, for example by adding <NUM> of N-Octadecylsuccinic acid anhydride per <NUM> to a mixture of acetophenone and acetone in equal volumes ("hydrophobic binder"). For conferring pH sensing properties to sections of the 3D printed object according to this example, a printing liquid comprising Methyl Orange was used. This printing liquid was produced by preparing a saturated solution of Methyl Orange in <NUM> of MilliQ™ water, which was subsequently mixed with <NUM> of Glycerol and 250µL of Surfactant (Surfynol <NUM>) ("pH indicator printing liquid").

Preparation of the digital model guiding the 3D printing: A digital 3D representation of a <NUM> x <NUM> x <NUM> plate containing a 3D flow channel with reactive areas was created using AutoCAD™ and exported as. Then, sections of said 3D representation were colored using Blender™.

org) as follows: white = hydrophilic section, such as hydrophilic channel or chamber, yellow (grey contour line in <FIG>, left picture) = section destined to comprise the pH indicator (Methyl Orange), black = hydrophobic sections. The obtained file was subsequently saved with a. wrl extension. Cyan text (light grey letters "PLACE SAMPLE HERE", RED: ACIDIC", "YELLOW: BASIC" in <FIG> left picture) was inputted onto device top face (as basic instruction for use) using 3D Software.

Printer setup: 3D printer (Projet™ <NUM> pro) is loaded with PMMA powder. The Clear printhead of this system was replaced with a custom printhead loaded with said printing liquid comprising a binder without hydrophobic agent ("hydrophilic binder", to be deposited on a predetermined "hydrophilic area"). This printhead was used for depositing the hydrophilic binder on the sections of the powder bed corresponding to the sections in the 3D digital representations indicated in white (<FIG>, white area in left picture and hydrophilic portion of the device in the right picture).

The Black binder printhead was replaced was replaced with a custom printhead loaded with a printing liquid comprising a binder combined with an hydrophobic agent ("hydrophobic binder", to be deposited on the predetermined "hydrophobic area"). This printhead was used for depositing the hydrophobic binder on the sections of the powder bed corresponding to the sections in the 3D digital representations indicated in black (<FIG>, left panel).

The Yellow binder printhead was replaced with custom printhead loaded with said printing liquid comprising a printing liquid comprising the pH indicator Methyl Orange ("pH indicator printing liquid", to be deposited on the predetermined "reactive area"). This printhead was used for depositing the pH indicator printing liquid on the sections of the powder bed corresponding to the sections in the 3D digital representations indicated in <FIG>, right panel.

The Cyan binder printhead was kept unchanged (prints blue commercial aqueous printing liquid).

Printing parameters were: <NUM> as layer thickness of powder material and <NUM>% as binder saturation.

3D printing process: Following the start of the printing process, the 3D printing device selectively deposits the respective printing liquids onto the powder bed corresponding to the printing instructions contained in the 3D digital representation: (a) Sections on which the hydrophilic binder is deposited, provide the hydrophilic areas which allow hydrophilic fluid sample transport; (b) Sections on which the pH indicator printing liquid is deposited provide pH sensitive reactive zones ("reactive area") of the object, and may comprise unbound powder, which allow hydrophilic fluid sample transport; and (c) Sections bound with the hydrophobic binder enclosing and/or delineating said hydrophilic and pH sensitive zones ("hydrophobic area") which do not allow hydrophilic fluid sample transport.

Post-processing: After termination of the drying step, the part is manually removed from the powder bed, brushed for removing last traces of powder. Thereafter the printed object is ready for use (<FIG>, right picture).

Claim 1:
A method for three dimensional printing of a porous device for liquid handling comprising hydrophobic sections delineating one or more interconnected chambers and/or channels enabling the capillary transport of hydrophilic fluids;
said method, which is a method without sintering, comprising the steps of:
a) Providing a print device comprising :
- means for spreading a dry powder material on a powder bed surface,
- one or more depositing outlets for depositing at least one printing liquid on said powder bed,
- means for repositioning said depositing outlets with respect to the surface of said powder bed, wherein said printing device allows for selectively depositing one or more printing liquids on the powder bed and,
- at least two printing liquids wherein at least one printing liquid comprises a hydrophobizing agent, and at least one printing liquid comprises a binder agent suitable for binding powder material ;
b) spreading a layer of dry powder of a particulate material over said powder plate in order to create a powder bed;
c) depositing one or more of said printing liquids comprising an agent suitable for binding powder material at predetermined positions on said powder bed, thereby binding the powder particles and depositing one or more of said printing liquids comprising a hydrophobizing agent at predetermined positions on said powder bed, thereby providing hydrophobic sections on the powder particles,
d) lowering the powder bed and spreading an additional layer of dry powder of the particulate material, wherein the dry powder is applied on top of both hydrophobic regions and hydrophilic regions of the underlying layer obtained in c);
e) repeating steps (c) and (d) until the object is formed, whereby at each repetition of step (c) an additional layer of the porous object is formed and wherein at least part of the powder material of the powder bed is bound to bound material of the layer formed in the previous step (c),
wherein said binding of layers results from the directed depositing of said printing liquid comprising said binder agent, and wherein the successive depositing of the one or more printing liquids comprising the hydrophobising agent results in delineating one or more interconnected chambers and/or channels within the hydrophobic sections wherein the chambers and/or channels are filled with hydrophilic particulate material enabling the capillary transport of hydrophilic fluids in between the hydrophilic particulate material.