Monoliths

Abstract

The present invention relates to a monolith for processing fluid samples, and methods of making and using the monolith. The monolith can contain certain monomers or combinations of monomers that can be polymerized to give a polymeric monolith that can efficiently self-wick fluid. The self-wicking polymeric monolith can be used as a convenient tool for point of care/on site diagnostics and analytics. The monolith is easily stored and transported, comparatively cost-efficient to make, permits good detection of analyte molecules and is readily functionalizable by impregnation of and/or covalently grafting additional chemical moieties to either the whole monolith or in zones.

1 - 20 . (canceled) 
     
     
         21 . A method of fabricating a self-wicking monolith for processing a fluid sample, the method comprising:
 providing at least one hydrophilic monomer and at least one linker monomer, the at least one linker monomer having two polymerizable groups spaced apart by a linker comprising at least one —C(R) 2 O— group; wherein each R is individually a hydrogen or an organic group;   optionally wherein at least one further monomer is provided;   obtaining a polymerizable composition by combining the at least one hydrophilic monomer and the at least one linker monomer in a porogenic solvent;   polymerizing the polymerizable composition to form the self-wicking monolith;   obtaining the self-wicking monolith as a polymeric matrix free of the porogenic solvent and free of any unpolymerized monomers: and   forming an amplification zone configured to facilitate isothermal amplification of target nucleic acid sequences or whole-genome amplification in the fluid sample.   
     
     
         22 . The method of  claim 21 , wherein each of the polymerizable groups of the at least one linker monomer comprises a vinylic moiety;
 and/or wherein the at least one linker is selected from the group consisting of:
 —O—CH 2 —CH 2 —O—; 
 (—O—CH 2 —CH 2 —) n —O—, wherein n is selected from 2, 3, 4, or 5; 
 —O—CH 2 —CH(OH)—CH 2 —O—; 
 —O—CH 2 —CH(OH)—CH 2 —CH(OH)—CH 2 —CH(OH)—CH 2 —O—; and 
 —OCH 2 —C(CH 2 O---)(CH 2 CH 3 )—CH 2 O—; 
   
       and/or wherein the at least one linker monomer is selected from the group consisting of: ethyleneglycol diacrylate, ethyleneglycol dimethacrylate, tetra(ethylene glycol) dimethacrylate, tetra(ethylene glycol) diacrylate, and di(ethylene glycol) dimethacrylate. 
     
     
         23 . The method of  claim 21 , wherein the at least one hydrophilic monomer is an acrylate or methacrylate; and optionally wherein the at least one hydrophilic monomer is: 2 hydroxyethyl methacrylate, 2-hydroxyacrylate, 2 hydroxyethyl acrylate, 2 hydroxypropyl methacrylate or 2 hydroxypropyl acrylate. 
     
     
         24 . The method of  claim 21 , wherein the at least one further monomer is functionalized and bears a moiety selected from the group consisting of:
 a chemically reactive group suitable for reaction with a reactive group of a graftable compound to covalently graft the graftable compound to the monolith;   a pH sensitive group;   a group suitable for direct immobilisation of an analyte;   a dye;   a fluorophore;   a chromophore;   a quencher;   an immobilised protein; and   immobilised natural or artificial nucleic acid molecules;   optionally wherein the at least one further functionalized monomer comprises at least one of the following side chains or groups: amino, carboxyl, polyethylene glycol, alkyl, maleimide, succinimide, acyl halide, sulfhydryl or azide;   optionally wherein the at least one further functionalized monomer is an amino methacrylate, an amino acrylate, acrylic acid or methacrylic acid; and   optionally wherein the total content of the at least one further functionalized monomer is 1-3% (v/v) of the total monomer content of the polymerizable composition.   
     
     
         25 . The method of  claim 21 , wherein:
 the porogenic solvent is able to dissolve solid monomers, or wherein the porogenic solvent is miscible with liquid monomers; and   the porogenic solvent is selected such that the at least one linker monomer forms clusters and precipitates from the porogenic solvent at an early point in polymerization;   and/or wherein the porogenic solvent is selected from the group consisting of:
 a binary mixture containing an alkane and an alcohol; 
 a binary mixture containing an aromatic solvent and an alcohol; 
 a binary mixture containing an alcohol and a diol; 
 a binary mixture containing an alcohol and water; 
 a ternary mixture containing an alcohol, a diol and water; and 
 a mixture containing at least 10% (v/v) surfactant. 
   
     
     
         26 . The method of  claim 21 , wherein more than one composition containing the at least one hydrophilic monomer and the at least one linker monomer in the porogenic solvent is provided, wherein two of the compositions vary in at least one of:
 the at least one hydrophilic monomer and/or the at least one linker monomer identity;   total non-linker monomer to the at least one linker monomer ratio;   the porogenic solvent;   the concentration of the at least one hydrophilic monomer, the at least one linker monomer, and the further monomer, if present, in the solution;   the presence and identity of the at least one further monomer; or   the presence and identity of an initiator;   
       the method further comprising a step of providing the more than one compositions at different locations within a mold prior to polymerization such that the self-wicking monolith comprises a plurality of zones, wherein different zones have different wicking properties and/or chemical properties. 
     
     
         27 . The method of  claim 21 , wherein the monolith is further modified wherein the modification comprises: chemical hydrolysis, covalent grafting of a graftable compound to the monolith, or impregnation of the monolith with one or more components. 
     
     
         28 . The method of  claim 21 , further comprising forming a plurality of zones, wherein different zones have different wicking properties and/or chemical properties, and at least two zones are ordered sequentially or in parallel along an intended wicking direction of the monolith. 
     
     
         29 . The method of  claim 28 , wherein at least one of the zones is a clean-up zone configured to perform at least one of the following:
 mechanical entrapment of a component in the fluid sample to retard or prevent movement of the component through a monolith during wicking;   affinity entrapment of a component in the fluid sample to retard or prevent movement of the component through a monolith during wicking;   facilitate one or more of polynucleotide release via disruption of somatic cells, viruses, bacteria, or fungi; cytosol release through cell lysis, or   facilitate release of an analyte through disruption of masking effects in a sample matrix of the fluid sample.   
     
     
         30 . The method of  claim 28 , wherein at least one of the zones is a reverse transcription zone which is configured to facilitate transcription of RNA to cDNA. 
     
     
         31 . The method of  claim 30 , wherein the reverse transcription zone comprises a reverse transcriptase, wherein the reverse transcriptase is impregnated into the monolith or covalently grafted into the monolith. 
     
     
         32 . The method of  claim 28 , wherein at least one of the zones is an indication zone configured to facilitate detection of an analyte. 
     
     
         33 . The method of  claim 32 , wherein the indication zone includes a dye fluorophore or a chromophore and optionally a quencher. 
     
     
         34 . The method of  claim 32 , wherein the indication zone is configured to perform nucleic acid hybridization and signaling of DNA. 
     
     
         35 . The method of  claim 34 , wherein the nucleic acid hybridization and signaling of DNA comprises a nicking endonuclease (NESA) hybridization and signaling process. 
     
     
         36 . The method of  claim 35 , wherein the indication zone is configured to perform the nicking endonuclease (NESA) hybridization and signaling process by:
 impregnating the polymeric matrix with a DNA probe and/or covalently grafting a DNA probe to the polymeric matrix; and   impregnating the polymeric matrix with a nicking enzyme and/or covalently grafting a nicking enzyme to the polymeric matrix.   
     
     
         37 . The method of  claim 21 , wherein the isothermal amplification is selected from the group consisting of: strand displacement amplification (SDA), rolling circle amplification (RCA), recombinase/polymerase amplification (RPA), exponential amplification reaction (EAR), helicase dependent amplification (HDA) and loop mediated amplification (LAMP). 
     
     
         38 . The method of  claim 21 , wherein the forming an isothermal amplification zone comprises:
 impregnating and or covalently grafting into the polymeric matrix: a plurality of deoxynucleotide triphosphates, at least one primer, a polymerase and a buffer; and optionally: at least one stabilizer and/or at least one salt.   
     
     
         39 . The method of claim  18 , wherein the at least one primer comprises: a plurality of inner primers and a plurality of outer primers and optionally a plurality of loop primers, wherein the amplification zone is configured for loop mediated amplification (LAMP). 
     
     
         40 . The method of  claim 21 , wherein the obtaining the self-wicking monolith as a polymeric matrix comprises:
 washing away the porogenic solvent and any unpolymerized monomers; and   drying the self-wicking monolith.