WO2013072408A1 - Fibres cellulosiques à surface fonctionnalisée, procédé de fabrication associé et application associée - Google Patents

Fibres cellulosiques à surface fonctionnalisée, procédé de fabrication associé et application associée Download PDF

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WO2013072408A1
WO2013072408A1 PCT/EP2012/072706 EP2012072706W WO2013072408A1 WO 2013072408 A1 WO2013072408 A1 WO 2013072408A1 EP 2012072706 W EP2012072706 W EP 2012072706W WO 2013072408 A1 WO2013072408 A1 WO 2013072408A1
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functionalized
polynucleotide
polynucleotides
group
functional group
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PCT/EP2012/072706
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Patrik STÅHL
Harry Brumer
Joakim Lundeberg
Ana Catarina DE ARAÚJO SILVA
Yajing SONG
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Swetree Technologies Ab
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    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
    • D06M13/322—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing nitrogen
    • D06M13/402—Amides imides, sulfamic acids
    • D06M13/425—Carbamic or thiocarbamic acids or derivatives thereof, e.g. urethanes
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
    • D06M13/322—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing nitrogen
    • D06M13/402—Amides imides, sulfamic acids
    • D06M13/432—Urea, thiourea or derivatives thereof, e.g. biurets; Urea-inclusion compounds; Dicyanamides; Carbodiimides; Guanidines, e.g. dicyandiamides
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/04—Vegetal fibres
    • D06M2101/06—Vegetal fibres cellulosic
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2400/00—Specific information on the treatment or the process itself not provided in D06M23/00-D06M23/18
    • D06M2400/01—Creating covalent bondings between the treating agent and the fibre

Definitions

  • the present invention related to surface functionalized cellulosic fibres, as well as methods of manufacture for such fibres, product in which comprise such fibre and uses/methods related to the use of such fibres.
  • paper-based materials Since the first production of paper sheets from cellulosic plant fibres centuries ago, paper- based materials continue to be harnessed in a growing number of applications, motivated, in part, by a low cost, high strength-to-weight ratio, and inherent resource renewability. Beyond traditional uses as a substrate for writing and the graphic arts, paper and
  • bioactive paper i.e., advanced, cellulose-based functional materials that have the capacity to bind, detect, and/or deactivate biological substances, ranging from small molecules, proteins, carbohydrates, and nucleic acids, to whole organisms such as fungi, bacteria and viruses.
  • bioactive papers including environmental remediation, security applications, "smart” (i.e., indicator) packaging, pathogen filters, and, not least, medical diagnostics.
  • petrochemical-derived plastics (beyond weight or environmental concerns) is the intrinsic porosity of the sheet structure, which facilitates chromatographic separations and inexpensive microfluidics devices based on capillary flow.
  • the functionality of paper-based diagnostics can be further elaborated by printing, coating, or impregnation technologies, as well as covalent chemical modification.
  • paper-based assay platforms have been available since the mid-twentieth century; perhaps the most well-known example of which is the pregnancy test stick, an immunochromatographic assay based upon a lateral-flow device. Recent times have however witnessed a resurgence in the development of paper- based assays, using paper either as a separation medium or as a sample capture and transport medium in "dipstick" analyses.
  • DNA-based diagnostic tests which are at the same time simple, affordable, sensitive, specific, rapid, and robust remains a major challenge in many application areas.
  • the last decade has witnessed a significant increase in interest for new and improved deoxyribonucleic acid (DNA)-based diagnostic tests, and there presently exists significant scope to further expand the use of cellulose-based paper substrates for the specific capture and detection of DNA originating from clinical or forensic samples.
  • DNA- functionalized glass surfaces have traditionally been powerful tools for the hybridization- based isolation, detection, and analysis of specific DNA sequences.
  • hour-long hybridization times and detection methods requiring expensive and bulky equipment have imposed limitations on the broad commercial application of glass-supported DNA assays, especially in field diagnostics, including those in resource-poor regions.
  • FIG. 1 Schematic illustration of the methodology of activation of cellulose surfaces with PDITC for DNA immobilization and subsequent rapid hybridization with complementary DNA target.
  • Three types of cellulose surfaces were evaluated: filter paper, filter paper with XG and filter paper with amino-functionalized XG.
  • FIG. 1 A. Image of fluorescently labeled filter paper surfaces after hybridization, analyzed with FujiFilm luminescent Image Analyser. All papers shown above were reacted with 15.6 mM PDITC either in DMF or DMSO: a) FP/PDITC in DMF; b) FP/PDITC in DMSO; c) FP/XG- PDITC in DMF; d) FP/XG-PDITC in DMSO; e) FP/XG-NH 2 -PDITC in DMF; f) FP/XG-NH 2 -PDITC in DMSO; B. Cartoon representation of the filter paper spotting.
  • FIG. 3 The illustration shows the fluorescence intensity as a function of PDITC
  • FIG. 4 Sample detection using activated filter papers with FP/XG-NH 2 a. Schematic of the robotically printed detection array. The immobilized oligonuleotides are complementary to (Tl) Dog Mitochondrial amplicon, (T2) Dog Genomic amplicon, (T3) Human Mitochondrial amplicon and (T4) HumanGenomic amplicon. (neg) is a negative control and (SI) is complementary to the positive control probe PS_1. b. Fluorescent detection of Cy3-labeled hybridized ssDNA sample. For visualization, a cutoff was set according to the mean signal of the maximum recorded background. A fluorescence of zero meant there was no recorded signal. Sample ssDNA matches its complementary surface probe correctly, c.
  • Graphs depicting the signal intensities from the four detected samples shows that intensities are similar among the samples and the corresponding positive controls (PTC) and that the background signal from the negative control (NTC) and unspecific hybridization to surface probes are minimal.
  • Figure 5. Overview of sample preparation through PCR and strand separation.
  • the forward primer is coupled to a Cy3 label
  • the reverse primer is fused with a sequence ID-tag (T_l to T_4 respectively), and coupled to a biotin moiety.
  • T_l to T_4 sequence ID-tag
  • biotinylated PCR product is bound to streptavidin-coated paramagnetic beads.
  • the Cy3- labeled forward strand is eluted with NaOH and used for downstream detection.
  • Figure 6 Standard curve for ninhydrin-assay using amino-functionalized xyloglucan (XG-NH 2 ).
  • Figure 7 Binding isotherm of amino-functionalized xyloglucan (XG-NH 2 ) onto cellulose.
  • polynucleotide is to be interpreted broadly as any polymer comprising nucleosides capable of specific hybridization with a polynucleotide with a natural (phosphodiester) backbone.
  • the term encompasses polynucleotides with phosphodiester backbones, polynucleotides with phosphorothioate backbones, peptide nucleic acids (PNAs) and locked nucleic acids (LNAs).
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the term encompasses polynucleotides comprising naturally occurring bases adenine, thymine, cytosine, guanine and uracil but also
  • polynucleotides comprising one base analogue, polynucleotides comprising more than one base analogue, and polynucleotides comprising solely base analogues.
  • hybridization means a specific binding interaction between two polynucleotides.
  • specific has the meaning that the interaction is dependent on the degree of sequence complementarity between the two interacting polynucleotides. It is well known in the field that the specificity of the interaction depends on the temperature, the length of the interacting polynucleotides, the sequence (e.g. CG-content) of the interacting
  • polynucleotides the degree of sequence complementarity between the interacting polynucleotides, type of polynucleotide, components of the medium in which the
  • polynucleotides interact (such as salt content and crowding agents), the concentration of the interacting polynucleotides as well as many other factors.
  • concentration of the interacting polynucleotides as well as many other factors.
  • polynucleotides of interest will specifically hybridize to each other but will not hybridize to other targets to an interfering degree for the purpose(s) of the analysis being performed.
  • hybridization in the context of the present invention is such that it is specific in stringent conditions.
  • a surface-functionalized cellulosic fibre characterized in that it comprises a group according to formula (A) below:
  • the surface-functionalized cellulosic fibre may be characterized in that it comprises a functional group denoted X covalently linked to a cellulose molecule as depicted in formula (I):
  • the surface functionalized cellulosic fibre may comprise a xyloglucan molecule bound to a cellulose molecule.
  • the xyloglucan molecule may comprise a functional group X covalently linked to it as depicted in formula (III): s
  • the functional group X may comprise a moiety suitable for covalent coupling or non- covalent binding of polynucleotides or functionalized polynucleotides.
  • the moiety may comprise an amine-reactive moiety.
  • the moiety may preferably be an isothiocyanate gro an isocyanate group, an epoxide group, an acrylate group, an acrylamide group, an N- hydroxysuccinimidyl ester group or an imidoester group.
  • the functional group X may be as depicted in formula (II):
  • the functional group X may comprise a polynucleotide.
  • the functional group X may be non- covalently bound to a polynucleotide.
  • the polynucleotide may be a single-stranded DNA molecule.
  • the fibre comprises at least 1 ⁇ g polynucleotide per gram of total dry weight.
  • porous material comprising a surface functionalized cellulosic fibre according to the first aspect.
  • the porous material may be hydrophilic.
  • a paper comprising a surface functionalized cellulosic fibre according to the first aspect.
  • the paper may be a filter paper sheet.
  • a method of surface-functionalizing cellulosic fibre comprising the step of contacting a cellulosic fibre with a bifunctional reagent comprising a isothiocyanate group and a second functional group denoted X, under such conditions that the isothiocyanate group reacts with the OH-groups of the cellulose comprised in the fibre, thereby forming a modified cellulose molecule as depicted in formula (I):
  • the cellulosic fibre of the fourth aspect may comprise a xyloglucan molecule bound to a cellulose molecule.
  • the xyloglucan molecule is amine-functionalized.
  • the functional group X may comprise a moiety suitable for covalent coupling or non- covalent binding of polynucleotides or functionalized polynucleotides.
  • the moiety may comprise an amine-reactive moiety.
  • the moiety may preferably be an isothiocyanate group, an isocyanate group, an epoxide group, an acrylate group, an acrylamide group, an N- hydroxysuccinimidyl ester group or an imidoester group.
  • the bifunctional reagent may be 1,4-phenylenediisothiocyanate (PDITC) present in a solvent.
  • PDITC 1,4-phenylenediisothiocyanate
  • the solvent may be dimethylformamide (DM F) or dimethyl sulfoxide (DMSO).
  • the PDITC may be present in a solvent at a concentration of 1-500 mM, preferably 2-10 mM .
  • the bifunctional reagent comprises PDITC dissolved in DMSO at a concentration of about 5 mM.
  • the method of the fourth aspect may comprise the further step of covalently coupling or non-covalently binding a polynucleotide to the functionalized cellulosic fibre.
  • the second functional group is amine-reactive and the polynucleotide is amine-functionalized.
  • the polynucleotide may be a single-stranded DNA molecule.
  • I n a sixth aspect, there is provided a use of a surface-functionalized cellulosic fibre, porous material or paper according to the first, second, third or fifth aspects in an analytical, diagnostic or forensic method.
  • a method for analysis of a polynucleotide analyte via hybridization to an immobilized capture polynucleotide comprising the steps of: a) providing a hydrophilic porous material comprising surface-functionalized cellulosic fibres according to the first aspect, wherein the fibres are
  • the analyte may be labelled by way of amplification in a PCR reaction using a labelled primer.
  • a primer used in said PCR reaction may comprise a sequence which can be used for hybridization with the target polynucleotide, or a complementary sequence thereof.
  • the detectable label is preferably biotin or a fluorescent label.
  • the biotin label is preferably detected (in step e) by using beads or particles comprising avidin or streptavidin on the surface.
  • the duration of step c) is preferably less than 10 minutes.
  • the analyte concentration is preferably less than 1 ⁇ .
  • the porous material of the seventh aspect is preferably filter paper.
  • microscope slides are typically activated toward coupling with amino-modified DNA via initial reaction with an aminoalkyltrialkoxysilane, such as 3- aminopropyltriethoxysilane (APTES), to install surface amino groups, followed by treatment with the amine-reactive, homobifunctional linker 1,4-phenylenediisothiocyanate (PDITC).
  • an aminoalkyltrialkoxysilane such as 3- aminopropyltriethoxysilane (APTES)
  • APTES 3- aminopropyltriethoxysilane
  • PDITC amine-reactive, homobifunctional linker 1,4-phenylenediisothiocyanate
  • PDITC-based cross-linking chemistry widely employed for glass supports, could be transferred to porous filter paper as a matrix for aminated ssDNA probe immobilization, following initial activation with amino-xyloglucan (XG-NH 2 ).
  • the present invention provides a surface-functionalized cellulosic fibre characterized in that it comprises a group as depicted in formula (A) below:
  • Such cellulosic fibre may be obtained by way of the teachings contained herein.
  • the modifying group may be coupled to cellulose molecules and/or to xyloglucan molecules bound to the cellulose molecules.
  • the surface-functionalized cellulosic fibre may be characterized in that it comprises a functional group denoted X covalently linked to a cellulose molecule as depicted in formula (I):
  • the product shown in formula (I) may be achieved by reacting a bi-functional reagent comprising an isothiocyanate group and a second functional group X with cellulose, by guidance of the teachings herein.
  • a bi-functional reagent is PDITC.
  • the cellulosic fibre may optionally comprise a xyloglucan molecule bound to it.
  • the xyloglucan molecule may comprise a functional group X covalently linked to it as depicted in formula (III)
  • Formulas (IV) and (V) further illustrate the modified xyloglucan molecules of formula (III).
  • Formula (IV) exemplifies amine-modified xyloglucan
  • formula (V) exemplifies unmodified xyloglucan having been modified in the manner of the invention.
  • formulas (la), (IV) and (V) are provided for illustration only and not to be interpreted literally or to be limiting.
  • some of the R-groups may have additional alternative structures different from the ones given, such as in cases where the cellulose or xyloglucan is further modified in some additional manner not subject of the present invention.
  • the formulas (la), (IV) and (V) are not to be taken to imply that each repeating unit depicted in the parenthesis is identical to each other with regard to the R-groups and their placement.
  • the groups denoted R merely illustrate potential positions for a modification according to the invention.
  • isothiocyanate reagent such as 1,4-phenylenediisothiocyanate, PDITC
  • PDITC 1,4-phenylenediisothiocyanate
  • an isothiocyanate reagent such as PDITC
  • the surface-functionalized cellulosic fibre of formula (A) comprising xyloglucan according to formula (III) can also be obtained by a method comprising a first step of coupling the functional group X to xyloglucan or amine-modified xyloglucan using an isothiocyanate reagent and a second step of contacting the thus obtained modified xyloglucan according to formula (III) with a cellulosic fibre under suitable conditions, whereby the xyloglucan binds to the cellulose comprised in the fibre.
  • the functional group X may comprise a moiety suitable for covalent coupling or non- covalent binding of polynucleotides or functionalized polynucleotides, such as aminated polynucleotides.
  • Said moiety may comprise an amine-reactive moiety, such as an isothiocyanate group.
  • the moiety may preferably be an isothiocyanate group, an isocyanate group, an epoxide group, an acrylate group, an acrylamide group, an N-hydroxysuccinimidyl ester group or an imidoester group.
  • such functional group can be
  • any amine-containing molecule of interest may be readily covalently coupled to the cellulose or xyloglucan functionalised in this way.
  • a preferred example of an amine-containing molecule of interest is an aminated polynucleotide.
  • the functional group X comprises a polynucleotide which may be bound covalently or non-covalently.
  • the functional group X may comprise streptavidin, whereby a biotinylated polynucleotide may readily be bound to the functional group in a non-covalent manner.
  • said polynucleotide is single-stranded, which allows for a plurality of applications based on hybridization.
  • Said polynucleotide may have natural phosphodiester bonds or other bonds forming the backbone and may comprise base analogues (see Definitions).
  • said polynucleotide is a DNA molecule.
  • the surface-functionalized fibre may comprise at least 1 ⁇ g polynucleotide per gram of total dry weight, more preferably at least 5 ⁇ g per gram, even more preferably at least 10 ⁇ g per gram, yet more preferably at least 15 ⁇ g per gram, still more preferably at least 50 ⁇ g per gram and most preferably at least 100 ⁇ g per gram.
  • the fibre may comprise polynucleotide in a range formed by any combination of the above amounts, such as 1-100 ⁇ g per gram, 5-100 ⁇ g per gram, 5-50 ⁇ g per gram, 10- 50 ⁇ g per gram, 15-100 ⁇ g per gram, or 50-100 ⁇ g per gram.
  • the present invention discloses porous material comprising a surface functionalized cellulosic fibre of the first aspect.
  • Said porous material may be hydrophilic.
  • the porous hydrophilic material is such that it enables efficient capillary transport of aqueous liquids.
  • the present invention discloses paper comprising a surface functionalized cellulosic fibre of the first aspect.
  • Said paper may be filter paper, such as a filter paper sheet.
  • the present invention discloses a method of surface-functionalizing cellulosic fibre, comprising the step of contacting a cellulosic fibre with a bifunctional reagent comprising a isothiocyanate group and a second functional group denoted X, under such conditions that the isothiocyanate group reacts with the OH-groups of the cellulose comprised in the fibre, thereby forming a modified cellulose molecule as depicted in formula (I):
  • Said cellulosic fibre may comprise xyloglucan molecules bound to cellulose molecules.
  • a surface-functionalized cellulosic fibre comprising xyloglucan according to formula (III) can also be obtained by a method comprising a first step of contacting a xyloglucan or amine-modified xyloglucan with a bifunctional reagent comprising an isothiocyanate group and a second functional group denoted X, under such conditions that the isothiocyanate group reacts with the OH-groups of the xyloglucan and/or amine-groups of the amine-modified xyloglucan, and a second step of contacting the thus obtained modified xyloglucan (according to formula (III)) with a cellulosic fibre, whereby the xyloglucan binds to the cellulosic fibre.
  • the resulting product of said alternative method would not comprise modified cellulose fibres according to formula (I).
  • the functional group X may comprise a moiety suitable for covalent coupling or non- covalent binding of polynucleotides or functionalized polynucleotides.
  • said moiety may comprise an amine-reactive moiety, such as an isothiocyanate group.
  • the moiety may preferably be an isothiocyanate group, an isocyanate group, an epoxide group, an acrylate group, an acrylamide group, an N-hydroxysuccinimidyl ester group or an imidoester group.
  • Such groups can then readily be used to covalently couple any amine-containing molecules, such as aminated polynucleotides, to the cellulose or the xyloglucan and thereby the cellulosic fibre.
  • the functional group may comprise streptavidin, which can then readily be used to non-covalently bind any biotin-containing molecules, such as biotinylated polynucleotides to the cellulose.
  • Said bifunctional reagent may be PDITC, in which case the reacting step takes place in a solvent which is preferably DMF or DMSO.
  • the PDITC may be present in the solvent at a concentration of 1-500 mM, more preferably 2-10 mM.
  • the reagent PDITC may be dissolved in DMSO at a concentration of about 5 mM (e.g. 2-10 mM).
  • the method may comprise the further step of covalently coupling or non-covalently binding a polynucleotide to the functionalized cellulosic fibre.
  • the second functional group is amine-reactive and the polynucleotide is amine-functionalized.
  • Said polynucleotide is preferably single- stranded, such as single-stranded DNA.
  • a double stranded polynucleotide may be coupled or bound, optionally followed by separation of the strands.
  • a surface-functionalized cellulosic fibre obtainable by the method according to the fourth aspect. Methods of use for a surface-functionalized cellulosic fibre
  • the present invention discloses a use of a surface-functionalized cellulosic fibre, porous material or paper according to the first, second, third or fifth aspects aspect in an analytical, diagnostic or forensic method.
  • the present invention discloses a method for analysis of a
  • polynucleotide analyte via rapid hybridization to an immobilized target polynucleotide comprising the steps of: a) providing a hydrophilic porous material comprising surface-functionalized
  • the material comprises a target polynucleotide capable of hybridization with the analyte, preferably in stringent conditions;
  • the analyte may be labelled by way of having undergone amplification in a PCR reaction using a labelled primer.
  • At least one of the primers used in the PCR reaction may preferably comprise a sequence which can be used for hybridization with the target polynucleotide, or a complementary sequence thereof
  • Said detectable label may preferably be a fluorescent marker such as Cy3 or FITC, an enzymatic marker, a dye marker, biotin, a particle or a bead, more preferably biotin or a fluorescent marker.
  • Said particle or bead is preferably dyed or otherwise visible.
  • the biotin label is detected by using coloured beads or particles comprising avidin or streptavidin on the surface. An example of such a detection method is disclosed in PCT/EP2010/069617.
  • the duration of step c) is less than 30 minutes, more preferably less than 10 minutes, even more preferably less than 5 minutes, most preferably less than 30 seconds.
  • the analyte concentration is less than 1 ⁇ , more preferably less than 200 nM, most preferably less than 30 nM.
  • the porous material is preferably filter paper.
  • Example 1 Modification of cellulose surface chemistry followed by covalent
  • Whatman No.l filter paper was selected as a matrix due to low non-specific biomolecule adsorption, good aqueous flow characteristics, cost-effectiveness, and ready availability.
  • Filter papers bearing XG-NH 2 as well as control sheets bearing unmodified XG of the same molar mass, were initially prepared by adsorption over 24h at room temperature under orbital agitation.
  • An adsorption isotherm using a ninhydrin assay for primary amino groups indicated that saturation of the paper surface with XG-NH 2 was achieved at addition levels of 250 mg XG-NH 2 per gram of filter paper (24 h adsorption, Figures 5 and 6).
  • an addition level 50 mg XG-NH 2 per gram of filter paper (5% w/w) was selected to economize the use of the polysaccharide reagent.
  • the reaction of PDITC with aminoalkylsilane-modified glass surfaces is typically performed in organic solvents such as DMF or dichloromethane. Due to the highly polar, hydrophilic nature of the filter paper surfaces, the inventors were interested to explore polar solvents for PDITC activation of cellulose and XG-modified cellulose.
  • PDITC is, however, practically insoluble in water, thus, filter papers bearing XG-NH 2 (FP/XG-NH 2 ), unfunctionalized XG (FP/XG), and control filter papers (FP) were immersed and orbitally shaken in solutions of PDITC of increasing concentration up to 500 mM in ⁇ , ⁇ -dimethylformamide (DMF) or 250 mM in dimethylsulfoxide (DMSO). Subsequent array spotting of aminated ssDNA by hand pipetting, capping of unreacted surface thiocyanate groups with ethanolamine, and hybridization with a fluorescently (Cy3) labeled complementary strand under capillary flow conditions allowed the relative immobilization capacity of these papers to be quantified under functional assay conditions.
  • FP/XG-NH 2 unfunctionalized XG
  • FP control filter papers
  • FIG. 2 shows a representative set of paper strips produced by varying the surface preparation and solvent conditions. As highlighted by Figure 2, signal is clearly visible above the background in all cases, and the cross-reaction between the target and a second sequence probe immobilized on the surface is undetectable.
  • Figure 3 summarizes the quantitative data similarly obtained for range of PDITC concentrations in different solvents (DMSO and DMF).
  • FITC fluorescein isothiocyanate
  • Figure 4a shows the results after detection with each of the four amplicon samples on independent paper chips. Integrated signal intensities were comparable for similar amounts of sample, as shown in Figure 4c.
  • ⁇ , ⁇ -dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Sigma- Aldrich and dried over 4 A molecular sieves for at least 24 h prior to use.
  • Ultrapure water (18 ⁇ ) was obtained from a Milli-Q. purification system (Milipore) and was used in all experiments.
  • Xyloglucan (XG, w 4345 Da, w / n 1.2) and amine-functionalized xyloglucan (XG-NH 2 , 19 synthesized from the aforementioned XG by reductive amination) was obtained from SweTree Technologies (Stockholm, Sweden).
  • Whatman No. 1 filter paper sheets (7.5x10cm, total mass 630 mg) were each cut in half lengthwise immersed in 32 mL of an aqueous solution of XG or XG-NH 2 (32 mg) in a rectangular glass tray with a glass cover. The mixture was placed on an orbital shaker for 24 h at room temperature, after which time it was washed twice with water (2 x 60 mL) for 5 min and dried under a gentle stream of air. Under these conditions, 50% of the total amount of the XG-NH 2 was sorbed into cellulose at this concentration (cf. Figure 6). Activation of filter paper with PDITC
  • Oligonucleotide design Oligonucleotides for immobilization and detection were designed using software developed in-house, and ordered from the manufacturer (Sigma-Aldrich, St. Louis, USA & MWG, Ebersberg, Germany). Unique sequences for surface immobilized oligonucleotides T_l through T_4 (Table SI) were created through generating 30-mer randomized ID tag sequences and mapping against the human and dog genomes, and to each other. The top ranking sequences with maximum difference to each other and minimum cross-reactivity were selected. Sequences S_l, 0_2 and PS_1 (Table SI) were generated with less stringency and were used as test probes for surface chemistry optimization.
  • Array preparation DNA arrays were prepared on the surface of the activated filter paper chips through either manual or robotic printing.
  • Chips were treated by submersion in a pre-warmed (50°C) "blocking" solution of 0.1 M TRIS and 50 mM ethanolamine, pH 9.0, to quench unreacted isothiocyanate groups. Chips were then washed sequentially with deionized water (3 x 2 mL, 2 min), a pre-warmed (50°C) solution of 4x saline-sodium citrate (SSC) buffer containing 0.1% sodium dodecylsulfate (2 mL, 30 min), and deionized water (3 x 2 mL, 2 min), followed by drying in air.
  • SSC 4x saline-sodium citrate
  • Arrays of oligonucleotide probes were printed with an array printer (Nano-PlotterTM NP2.1, GeSiM, Germany) on FP/XG-NH 2 surfaces.
  • a pattern of eight arrays per filter paper chip (7.5 x 2.5cm) was prepared by plotting a solution containing 20 ⁇ oligonucleotide probe in 50mM sodium phosphate buffer, pH 8.5, respectively for each probe.
  • Six features were included in each array, consisting of four oligonucleotide ID_tag probes (T_l through T_4), one positive control (S_l), and one negative control (buffer only) ( Figure 4a). Following printing, chips were maintained in a humid atmosphere overnight, blocked with
  • Each 50 ⁇ PCR mixture contained 1U Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) , IX Platinum Taq PCR buffer (Invitrogen, Carlsbad, CA, USA), 0.2mM dNTPs, 1.5 mM MgCI 2 , 200 nM each of a Cy3-labeled forward primer and a biotin-labeled reverse primer, and ⁇ extracted sample DNA.
  • the reaction mixture was processed at 94°C for 5m; cycled 45 times through 94°C for 30 s, 58°C for 40 s, and 72°C for 60 s, and after the cyclic amplification final elongation step of 72°C for 10 min was performed.
  • the filter paper chips from the surface chemistry optimization were scanned in a Fujifilm Luminescent Image Analyzer and the signal intensity was measured using the associated software (Fujifilm, Tokyo, Japan).
  • Filter papers used in the dog/human sample detection assays were scanned in a Tecan LSTM Series Laser Scanner (Tecan, Switzerland) and the images were analyzed using the GenePix 5.1 software (Molecular Devices Corporation, USA).

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Abstract

La présente invention concerne une fibre cellulosique à surface fonctionnalisée, caractérisée en ce qu'un groupe fonctionnel noté X est lié de façon covalente à la cellulose avec une liaison ayant la structure : X-(NH)-(C=S)-0-molécule de cellulose, comprenant en outre facultativement du xyloglucane. L'invention concerne également des procédés de fabrication associés et des applications associées dans des procédés analytiques.
PCT/EP2012/072706 2011-11-15 2012-11-15 Fibres cellulosiques à surface fonctionnalisée, procédé de fabrication associé et application associée WO2013072408A1 (fr)

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WO2017123311A3 (fr) * 2015-11-03 2017-09-21 President And Fellows Of Harvard College Dispositif basé sur un substrat cellulosique
CN109196160A (zh) * 2016-06-03 2019-01-11 花王株式会社 血细胞凝集性纤维

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017123311A3 (fr) * 2015-11-03 2017-09-21 President And Fellows Of Harvard College Dispositif basé sur un substrat cellulosique
CN109196160A (zh) * 2016-06-03 2019-01-11 花王株式会社 血细胞凝集性纤维

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