WO2016041022A1 - Échafaudage de capteur - Google Patents

Échafaudage de capteur Download PDF

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Publication number
WO2016041022A1
WO2016041022A1 PCT/AU2015/050564 AU2015050564W WO2016041022A1 WO 2016041022 A1 WO2016041022 A1 WO 2016041022A1 AU 2015050564 W AU2015050564 W AU 2015050564W WO 2016041022 A1 WO2016041022 A1 WO 2016041022A1
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Prior art keywords
scaffold
polymer
metallic
particles
group
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PCT/AU2015/050564
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English (en)
Inventor
Matthew Cooper
Andrea Ranzoni
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The University Of Queensland
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Priority claimed from AU2014903750A external-priority patent/AU2014903750A0/en
Application filed by The University Of Queensland filed Critical The University Of Queensland
Publication of WO2016041022A1 publication Critical patent/WO2016041022A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/56—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing sulfur
    • C09K11/562—Chalcogenides
    • C09K11/565—Chalcogenides with zinc cadmium
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/88—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
    • C09K11/881—Chalcogenides
    • C09K11/883—Chalcogenides with zinc or cadmium
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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

Definitions

  • the present invention relates to sensors and in particular to a scaffold and a method for constructing a scaffold for a sensor for detecting a target molecule in a sample.
  • Detection of target molecules or analytes in biological samples is central to diagnostic monitoring of health and diseases. Key requirements of analyte detection are specificity and sensitivity, particularly when the target molecule or analyte is in a limiting amount or concentration in a biological sample.
  • detection agents such as antibodies which specifically interact with (i.e. bind or recognize) the analyte.
  • Sensitivity is typically provided by a label bound to the specific antibody, or to a secondary antibody which assists detection of relatively low levels of analyte.
  • biosensors which are analytical devices that combine a solid support sensor surface upon which the detection agents are immobilised, with a physicochemical detector.
  • ELISA enzyme-linked immunosorbent sandwich assay
  • the methods via which the detection agents are immobilised typically involve physical or covalent immobilisation of the detection agents to the sensor surface in a specific manner while minimising non-specific binding of undesirable materials (i.e. unwanted binding of proteins, cells, bacteria, and the like) through the use of biocompatible coatings.
  • undesirable materials i.e. unwanted binding of proteins, cells, bacteria, and the like
  • biocompatible coatings i.e. biocompatible coatings.
  • such methods often suffer from a number of limitations which significantly restrict their widespread application. Such limitations most notably include poor sensitivity due to an insufficient number of capture probes being immobilised on the sensor surface or poor access to the immobilised capture probes.
  • quantum dots which are fluorescent particles typically composed of semiconductor materials that bridge the gap between individual atoms and bulk semiconductor solids. Owing to this intermediate size, quantum dots possess unique properties unavailable in either individual atoms or bulk materials.
  • the small size of quantum dots means that the electronic characteristics (that is, the bandgap and emission wavelength of a quantum dot) may be tuned by simply adjusting its size, with smaller quantum dots emitting at shorter wavelengths.
  • UV ultraviolet
  • IR infrared
  • quantum dots can absorb and emit light very efficiently, allowing highly sensitive detection relative to conventionally used organic dyes and fluorescent proteins. Indeed, individual quantum dots are significantly brighter than organic dyes, thus enabling highly sensitive detection of analytes in low concentration. As well as having excellent fluorescence intensity, quantum dots also possess a photobleaching resistance ability making them ideally suited as labels for capture probes to detect specific target molecules or analytes in low concentration.
  • a method of constructing a scaffold for a sensor comprising the steps of: contacting a scaffold surface with a polymer, the polymer having a reactive functionality suitable for binding to each of (i) the scaffold surface; (ii) a metal or metal alloy; and (iii) a detection agent;
  • the scaffold surface may be a metallic or metal alloy surface, a ceramic surface, a silica surface or a polymeric surface.
  • the metallic surface may be a gold or silver plate or magnetic nanoparticle.
  • the silica surface may be colloidal silica or a surface of a silica-containing particle.
  • the polymeric surface may be a surface of a polymeric matrix, preferably of a polymer bead.
  • the contacting of the polymer with the plurality of metallic or metallic alloy particles may occur at the same time as or subsequent to the contacting of the scaffold surface with the polymer.
  • the contacting of the polymer with the plurality of metallic or metallic alloy particles occurs at the same time as the contacting of the scaffold surface with the polymer.
  • the reactive functionality suitable for binding to each of the scaffold surface, the metal or metal alloy and the detection agent may be the same or different.
  • the reactive functionality suitable for binding to the scaffold surface, the metal or metal alloy and the detection agent are the same functionality.
  • the reactive functionality suitable for binding to the scaffold surface, the metal or metal alloy and the detection agent may be a different functionality for one or more of the same.
  • the reactive functionality suitable for binding to the scaffold surface, the metal or metal alloy and the detection agent may be a chalcogen-containing functionality.
  • the reactive functionality comprises two bonded chalcogen atoms each of which may be independently selected from S, O, Se and Te, for example S-S.
  • the chalcogen atoms may have one or two oxygen atoms bonded thereto.
  • the terminal chalcogen atom is attached to an R group to form a leaving group.
  • the R group is selected such that the conjugate acid formed by the R group and attached chalcogen atom leaving the polymer has a pKa of less than 8, preferably less than 6 and more preferably less than 4.
  • HS-R could be the conjugate acid formed by the leaving group of an -S-S-R moiety.
  • the reactive functionality suitable for binding to each of the scaffold surface, the metal or metal alloy and the detection agent may be independently selected from a disulfide-containing functionality, a maleimide containing functionality and a functionality reactive with a thiol group.
  • the reactive functionality suitable for binding to the scaffold surface, the metal or metal alloy and the detection agent comprises -S-S-R wherein R is selected from an electron withdrawing group, aryl, heteroaryl and C1-C12 alkyl, each of which may be substituted.
  • substitution is with an electron withdrawing group such as halide, nitro, nitrile and carbonyl.
  • R is heteroaryl it may be selected from pyrrole, imidazole, pyrazole, pyridine, furan, thiophene, oxazole, thiazole, diazine, oxazine and thiazine.
  • R comprises a 2-pyridyl group.
  • the polymer may be a branched hydrophilic polymer.
  • the polymer may be a dendrimer or a polysaccharide, each of which may be optionally derivatised.
  • the polymer is selected from the group consisting of dextran, cellulose, agarose, sepharose, hyaluronic acid, agarose, nitrocellulose, polyvinylalcohol, partially hydrolysed polyvinylacetate and polymethylmethacrylate, carboxymethyl cellulose, carboxymethyl dextran and the like, any one of which may optionally be derivatised.
  • the polymer is 2-(pyridinyldithio)ethyl carbomoyi dextran (PDEC-dextran), PDEA-dextran or CMD-dextran.
  • PDEC-dextran 2-(pyridinyldithio)ethyl carbomoyi dextran
  • PDEA-dextran PDEA-dextran
  • CMD-dextran Polymers such as CMD-dextran may be appropriately modified to display the required functionality.
  • the metallic or metallic alloy particles are selected from the group consisting of gold particles, silver particles, platinum particles, iron particles and other magnetic particles, copper particles and metal-containing quantum dots.
  • the particles may be nanoparticles.
  • the metal-containing quantum dots are selected from the group consisting of a lll-V group compound, a ll-VI compound, and a IV group element.
  • the metal-containing quantum dots are selected from the group consisting of CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, CdSe/ZnS, ZnCdSe/ZnS, InP, InN, InAs, InGaP, GaP and GaAs.
  • the metal-containing quantum dots are organic metal- containing quantum dots.
  • the metallic or metallic alloy particles have an average particle size of 0.5 to 100 nm.
  • the method may further comprise the step of choosing the amount of the metallic or metallic alloy particles which are made available to the polymer to thereby control the thickness and/or density of the multi-layer polymer network which forms.
  • the method further comprises the step of attaching a fluorescent probe to the polymer.
  • the fluorescent probe may be attached to the reactive functionality, preferably via a thiol group.
  • the method further comprises the step of attaching a reporter agent, for example HRP enzyme, to the polymer
  • the method may further comprise, before step (a), the step of incorporating a plurality of metallic or metallic alloy particles within the polymer matrix.
  • the metallic or metallic alloy particles incorporated within the polymer matrix may be the same or different from the metallic or metallic alloy particles cross- linked within the multi-layer polymeric network. They may be selected from the same groups already described.
  • the metallic or metallic alloy particles incorporated within the polymer matrix may be magnetic particles, including iron particles.
  • the polymer matrix comprises both magnetic particles and a second population of particles selected from gold nanoparticles, silver nanoparticles and quantum dots.
  • the method further comprises, before the step of incorporating the metallic or metallic alloy particles within the polymer matrix, the step of swelling the polymer matrix in an organic solvent. This assists with incorporation of the metallic or metallic alloy particles therein.
  • the method may further comprise, prior to contacting the scaffold surface with the polymer, the step of adding a polymerisable reagent to the swollen polymer matrix with incorporated metallic or metallic alloy particles.
  • the method may further comprise the step of initiating polymerisation of the polymerisable reagent to form polymer strands located within the polymer matrix and forming part of the scaffold surface to thereby entrap the plurality of metallic or metallic alloy particles incorporated into the polymer matrix.
  • the polymerisable reagent has an anchoring group which, after the polymerisation of the polymerisable reagent, presents as part of the scaffold surface as a group reactive with the reactive functionality of the polymer.
  • the anchoring group of the polymerisable reagent is a thiol group.
  • the method is a method of constructing a scaffold for a sensor comprising the steps of:
  • a scaffold for a sensor comprising:
  • the scaffold surface, multi-layer polymeric network and metallic or metallic alloy particles are as described for the first aspect.
  • the scaffold surface is the surface of a polymer matrix
  • the scaffold may further comprise a plurality of metallic or metallic alloy particles within the polymer matrix.
  • the metallic or metallic alloy particles incorporated within the polymer matrix may be the same or different from the metallic or metallic alloy particles cross- linked within the multi-layer polymeric network.
  • the metallic or metallic alloy particles incorporated within the polymer matrix may be substantially physically entrapped therein by the presence of polymer strands formed from polymerisation of a polymerisable reagent.
  • the polymer strands formed by the polymerisable agent are preferably different from the polymer forming the multi-layer polymeric network.
  • the metallic or metallic alloy particles incorporated within the polymer matrix may comprise magnetic particles.
  • the multi-layer polymeric network may be bound to the scaffold surface, at least in part, via an anchoring group presented by the polymer strand formed from the polymerisable reagent, at least a portion of the polymer strand forming part of the scaffold surface.
  • a sensor for use in detecting a target molecule in a sample comprising:
  • a method of detecting a target molecule in a sample comprising the steps of:
  • the target molecule may be any molecule that is detectable by the detection agent.
  • Such target molecules may be any molecule that is capable of forming a complex or conjugate with the detection agent.
  • Non-limiting examples of target molecules include proteins inclusive of antibodies or antibody fragments, peptides, enzymes, epitopes and other antigens, antigen receptors, hormone receptors, growth factor receptors, cytokine receptors, hormones, growth factors or cytokines, small organic molecules inclusive of drugs and metabolites, carbohydrates, lipids, cells and cellular fractions, organelles or other components thereof, biological markers such as cell surface markers, nucleic acids such as single- or double stranded DNA and RNA, and derivatives, cooligomers and any fragments or combinations thereof.
  • Small molecules, ions, carbohydrates, whole cells, pathogens, bacteria (inclusive of Gram positive and Gram negative bacteria), viruses, protozoa, fungi and the like and/or molecular components thereof may also be, or comprise, a target molecules inclusive of antibodies or antibody
  • the detection agent may be any molecule that can be bound to the multi-layer polymeric network which is capable of forming a complex or conjugate with the target molecule.
  • the at least one detection agent include small molecules, ions, carbohydrates, whole cells, bacteria, viruses or molecular components thereof, lectins including WGA and ConA, fungi as well as proteins inclusive of antibodies or antibody fragments, peptides, enzymes, epitopes and other antigens, antigen receptors, hormone receptors, growth factor receptors, cytokine receptors, hormones, growth factors or cytokines, , cells and cellular fractions, organelles or other components thereof, biological markers such as cell surface markers, nucleic acids such as single- or double stranded DNA and RNA, and derivatives, cooligomers and any fragments or combinations thereof.
  • the detection agent may present a functional group for binding to the polymer of the multi-layer polymeric network.
  • the functional group may be naturally present on the detection agent or the detection agent may be modified to present the required group.
  • the detection agent presents a free thiol group to enable binding to the multi-layer polymeric network.
  • the detection agent and the target molecule may be "complementary binding partners".
  • an antibody and antigen e.g. an epitope
  • complementary binding partners could include complementary DNA strands, growth factors and growth factor receptors and enzymes and substrates, although without limitation thereto.
  • the sensor and method of detecting a target molecule may be useful for detecting target molecules in samples such as biological samples, diagnostic samples, food samples, air samples, water samples and the like.
  • samples such as biological samples, diagnostic samples, food samples, air samples, water samples and the like.
  • biological and/or diagnostic samples include tissue, organ and tumour biopsies, body fluids such as urine, blood, serum, cerebrospinal fluid, semen, tears and sputum, although without limitation thereto.
  • the detection agent is an antibody or antibody fragment.
  • the antibody or antibody fragment may be polyclonal, monoclonal or recombinant synthetic inclusive of single chain fragments such as scFvs, F(ab) and F(ab') 2 fragments, diabodies and triabodies, although without limitation thereto.
  • a preferred sensor may be suitable for use in an immunoassay.
  • One particular embodiment of the method of the fourth aspect includes:
  • the target molecule is bound to another antibody or antibody fragment prior to step (a).
  • said another antibody or antibody fragment may be coupled or immobilized to a surface of a vessel, such as a well of a microwell plate.
  • the method may include the step of exposing the sensor comprising metal nanoparticles to a second population of particles comprising incorporated quantum dots.
  • the metal nanoparticles are preferably gold or silver nanoparticles within the multi-layer polymeric network.
  • the two populations of particles are in a sample with the target molecule and the reduction in fluorescence of the quantum dots is measured as an output.
  • Fig. 1 shows (i) a standard curve of fluorescence intensity (a.u.) versus quantum dot concentration (mol) for a supernatant of a first washing step (in 5:95 toluene-butanol) following washing of a suspension of swollen polymer microspheres loaded with quantum dots according to one preferred embodiment of the present invention, and (ii) a corresponding chart to estimate the incorporation efficiency of quantum dots within the loaded microspheres;
  • Fig. 2 shows (i) a standard curve of absorbance (at 412 nm) versus sulfhydryl group concentration (mol), and (ii) a corresponding chart to quantify the density of sulfhydryl groups on the surface of five different batches of polymer bead loaded with quantum dots of Fig. 1 ;
  • Fig. 3 shows (i) a standard curve of fluorescence intensity (a.u.) versus quantum dot concentration (pmol) for a supernatant of a first washing step (in 5:95 toluene-butanol) following washing of a suspension of polymer beads after incubation in 2-(pyridinyldithio)ethyl carbomoyl dextran (PDEC-dextran) in the presence of a plurality of additional quantum dots to produce a multi-layer polymeric network with cross-linked additional quantum dots, and (ii) a corresponding chart to estimate the incorporation efficiency of additional quantum dots within the multi-layer polymeric network;
  • a first washing step in 5:95 toluene-butanol
  • PDEC-dextran 2-(pyridinyldithio)ethyl carbomoyl dextran
  • Fig. 4 shows (i) a standard curve of absorbance (at 562 nm) versus immunoglobulin (IgG) concentration ( g/mL), and (ii) a corresponding chart to estimate the density of antibody fragments (mFAbs) immobilized on the surfaces of scaffolds prepared according to one preferred embodiments of the present invention; and (iii) a chart of fluorescence intensity (a.u.) versus logio quantum dot concentration (ng/mL) showing a comparison of a scaffold of the present invention with functionalised commercial particles (Life Tech. F8811 ) with the same antibody pair used in each experiment;
  • Fig. 5 shows a chart of fluorescence intensity (a.u.) versus storage time (days) which provides a comparison of the stability of a batch of scaffolds constructed according to the present invention against batches of two reference scaffolds;
  • Fig. 6 a) - c) is a series of graphs showing the progressive increase in the fluorescence intensity due to a) increase in HRP activity with an increase in number of polymer layers in the multi-layer polymer network b) increase in fluorescence intensity with increase in number of polymer layers in the multi-layer polymer network c) increase in fluorescence intensity with increase in QDs incorporated due to increase in number of polymer layers in the multi-layer polymer network;
  • Fig. 7 a) - c) is a series of graphs showing dose response curves for the detection of Myoglobin using a) HY_QD, b) HY_FP and c) HY_HRP particles;
  • Fig. 8 is a diagrammatic representation of the polymer bioconjugation chemistry for experiments on the attachment of lectins to substrate particles;
  • Fig. 9 is a graphical representation of the BCA protein quantification standard curve with comparison of protein content achieved on scaffolds using direct (triangle) and multi-layer polymeric network (square) coupling of WGA;
  • Fig. 10 is a graphical demonstration of the bacterial capture from undiluted platelets
  • Fig. 1 1 is a graphical representation of the results of non-specific binding experiments, with negative controls, in platelets;
  • Fig. 12 is a graphical representation of the detection limit for S.Aureus in platelets using MCC174 blue beads functionalized with a CMD-dextran multilayer polymeric network;
  • Fig. 13 is a graphical representation of the decrease in fluorescence of quantum dots with the use of either gold or silver nanoparticles after (a) 1 hour and (b) 2 hours;
  • Fig. 14 is a graphical representation of the decrease in fluorescence of quantum dots with the use of varying amounts of silver nanoparticles.
  • the present invention is predicated, at least in part, on the finding that a polymer which has a functionality capable of reacting with metallic or metallic alloy particles can be used to form the basis of a scaffold for a sensor by binding the polymer to a substrate surface of the proposed scaffold and allowing the polymer to crosslink with a plurality of the metallic or metallic alloy particles to generate a three dimensional multi-layer polymeric network with the particles bonded in place therein.
  • the polymer also has a functionality which allows the subsequent binding of a detection agent, such as an antibody or antibody fragment, to form the sensor.
  • this multi-layer polymeric network provides great advantages in operation compared with the formation of a simple monolayer of polymer or other species displaying a detection agent, including that it substantially enhances the reactive polymer surface area available for anchoring of detection agents.
  • This has the beneficial knock on effect that the greater density of detection agent, for example an antibody, on the scaffold will greatly improve capture kinetics thereby providing for improved sensitivity and increased speed during detection.
  • the multi-layer polymeric network also has the beneficial effect of greatly reducing nonspecific binding to the substrate.
  • the fixing of the metallic or metallic alloy particles within the multi- layer polymeric network reduces the level of quenching that is observed compared to the situation when metallic or metallic alloy particles are only present within the body the scaffold substrate, such as within the matrix of a polymer bead or microsphere.
  • the polymer matrix of the polymer bead can reduce the excitation signal observed during use of the sensor. Surprisingly, this problem is substantially reduced or avoided when the metallic or metallic alloy particles are incorporated within the multilayer polymeric network and therefore distanced from the polymeric matrix of the bead resulting in superior brightness being observed.
  • the brightness of the emitted signal can be greatly increased by entrapping metallic or metallic alloy particles both within the polymer matrix of the polymer bead substrate and also within the cross-linked multilayer polymeric network thereby greatly amplifying the response from the biosensor.
  • the invention has been developed primarily for use in sensing and detecting analytes of interest in biological samples and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.
  • multi-layer polymeric network signifies that the polymer structure referred to extends beyond a single layer (monolayer) on the scaffold surface. That is, the polymer structure referred to will have a complex 3- dimensional structure due to crosslinking with a plurality of metallic or metallic alloy particles comprising a plurality of layers of the polymer extending away from the scaffold surface to which only the lowermost polymers are actually directly physically anchored or bound to. Therefore, the multi-layer polymeric network comprises multiple interconnected polymers in extending between the substrate surface and the outer extent of the formed polymeric scaffold.
  • the scaffold surface may be a surface of a substrate such as a polymer bead, ceramic particle, silica-containing particle or colloidal silica, metal, such as a gold or silver electrode plate, or other metal or metal alloy structure.
  • a substrate such as a polymer bead, ceramic particle, silica-containing particle or colloidal silica, metal, such as a gold or silver electrode plate, or other metal or metal alloy structure.
  • the nature of the substrate, and hence the scaffold surface is not particularly limited and potentially any surface which can bind, or be coated, functionalised or modified in any manner to bind, the polymer forming the multi-layer polymeric network may be suitable.
  • a wide range of such substrates and scaffold surfaces are already in use commercially and would be known to the person skilled in the art.
  • any functionalised surface presenting, for example -NH 2 , -SH, -OH, - COOH and similar reactive moieties may be suitable.
  • an APTES or MPTS modified ELISA plate may be a suitable option as a substrate and hence scaffold surface.
  • the shape or form which the substrate takes is not particularly limited.
  • the only requirement is that the polymer to form the multilayer polymeric network can bind itself to the scaffold surface of that substrate. This may be achieved by a natural reactivity with the surface, for example the reactivity of a disulfide moiety attached to a good leaving group with a metallic or metallic alloy surface, or by modification of the surface, for example thiol modification of a polymer bead functionality or introduction into such a bead of a thiol-containing polymer to thereby react with the same disulfide moiety. Such a modification to introduce the required functionality is within the present use of the term "scaffold surface".
  • the term "scaffold surface" as used herein includes situations wherein a further polymer presenting the functionality is added to the polymeric matrix and the polymer to form the multi-layer polymeric matrix can access that functionality whether or not it physically sits at the surface of the substrate or not. Such a situation is explicitly considered to present the functionality on or as forming a component of the "scaffold surface".
  • the nature of that substrate may be selected, in certain embodiments, on the basis of its being capable of swelling to a reasonable size when suspended in a suitable solvent or solvent system to then receive a plurality of metallic or metallic alloy particles, such as quantum dots, within the pores of the swollen polymer matrix.
  • metallic or metallic alloy particles such as quantum dots
  • Such polymer substrates can be purchased commercially in various sizes and with various functional groups on their surface.
  • polyethylene-based microspheres, polystyrene-based microspheres and latex microspheres are common, but the present scaffold is not limited thereto.
  • a preferred solvent or solvent system comprises an organic component to not only induce the swelling of the polymer matrix, for example latex microspheres, but also assists in preventing aggregation of the latex microspheres.
  • protic solvents were useful.
  • examples of such solvents may include methanol, ethanol, isopropanol and butanol, which all successfully swelled the microspheres to a sufficient size, with the latter solvent demonstrating the best results.
  • the solvent preference may change with changing nature of the substrate polymer. Information on the swelling characteristics of various solid-phase particles is readily available in commercial catalogues and the like.
  • this embodiment comprises the step of incorporating a plurality of metallic or metallic alloy particles, such as quantum dots, within the pores of the polymer matrix of the swollen substrate, for example latex microspheres, to produce a plurality of substrate particles loaded with quantum dots, referred to herein as the loaded substrate.
  • a plurality of metallic or metallic alloy particles such as quantum dots
  • a polymerisable reagent is optionally added to the solution of loaded substrates.
  • the polymerisable reagent may, in one embodiment, be mercaptopropyltrimethoxysilane (MPTS) which in the presence of a trace amount of water causes the MPTS to polymerise inside the pores of the polymeric matrix of the loaded substrates to form polymer strands and complete the entrapment of the quantum dots within the loaded substrates.
  • MPTS mercaptopropyltrimethoxysilane
  • This step not only completes the entrapment process, it also presents a plurality of sulfhydryl groups, from the polymerised MPTS, to form part of the scaffold surface of the loaded substrate.
  • the role of the polymerised MPTS is essentially a physical one whereby the formed polymer strands are physically entangled or engaged with the polymer matrix of the substrate, such as the latex microsphere. This effect could be achieved in a number of alternative ways.
  • the inclusion of the MPTS also provides a thiol anchoring group which effectively forms part of the scaffold surface and so provides a functionality to react with a complimentary functionality of the polymer used to form the multi-layer polymeric network, but this may not be necessary if such a functionality is inherently provided by the scaffold surface or the surface is otherwise modified to present such reactive functionality.
  • polymerisable reagent will therefore depend on the anchoring group required, if any, to be located at the scaffold surface by that reagent once polymerisation is complete.
  • the polymerisable reagent will present at least two alkoxy or aryloxy groups to allow polymerisation upon the addition of small amounts of water or aqueous acid.
  • the polymerisable reagent comprises two or more Ci to C 12 , preferably Ci to Ce alkoxy groups.
  • the polymerisable reagent further comprises at least one group capable of reacting with a disulphide functionality.
  • the polymerisable reagent in addition to the alkoxy or aryloxy groups described above, further comprises a thiol anchoring group.
  • the scaffold substrate and the metallic or metal lic alloy particles used in the preferred embodiments of the present invention will depend largely on the specific requirements of the applications for which the scaffold is intended. This is particularly relevant in the case of quantum dots as the electronic characteristics of a quantum dot are closely related to its size and shape.
  • the bandgap in a quantum dot which determines the frequency range of emitted light, is inversely related to its size.
  • the frequency of emitted light increases as the size of the quantum dot decreases. Consequently, the color of emitted light shifts from red to blue when the size of the quantum dot is made smaller. This allows the excitation and emission of the quantum dots to be highly tunable.
  • the metal lic or metal lic alloy particles may be in the nature of gold, silver, platinum, copper or iron particles or may be metal-containing quantum dots.
  • quantum dots these can be selected from the group consisting of an ll l-V group compound, an ll-VI compound, or an IV group element (which are referred hereon in as an lll-V group quantum dot, an l l-VI quantum dot, or an IV group quantum dot, respectively).
  • Specific examples thereof include, but are not limited to, and may be selected from the following: CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, CdSe/ZnS, ZnCdSe/ZnS, InP, InN, InAs, InGaP, GaP and GaAs.
  • CdSe/ZnS hydrophobic quantum dots (PL-QD-O-590, as commercially supplied by PlasmaChem Tech GmbH).
  • the average size of the CdSe core of these quantum dots may vary between about 2 nm and about 10 nm, depending on the desired emission maximum required for the scaffold.
  • PlasmaChem Tech indicate on their website that a CdSe core diameter of 2.5 nm has an emission maximum of 530 nm, while a CdSe core diameter of 6.7 nm has an emission maximum of 650 nm.
  • the website also indicates that the ZnS outer shell surrounding the core has a thickness of between about 0.5 nm and 0.7 nm.
  • [001 16] it may be desirable to have magnetic particles incorporated into the substrate or multi-layer polymeric network. It may further be beneficial to have at least a second population of metallic or metallic alloy particles which provide alternative properties including better formation of the multi-layer polymeric network or fluorescence. For example, in one non-limiting embodiment, it may be desirable to have magnetic particles incorporated within the substrate and to optionally have quantum dots also incorporated. The multi-layer polymeric network generated on the surface of the substrate may then employ either quantum dots or metal particles, such as gold or silver particles, to form the scaffold.
  • quantum dots or metal particles such as gold or silver particles
  • the polymer can be any type of polymer that comprises a plurality of functional groups that can react with the plurality of complimentary groups on the scaffold surface.
  • the polymer is a branched hydrophilic polymer.
  • the polymer is capable of binding to a functional group present on the surface of the substrate.
  • This functional group may be part of the substrate structure such as, for example, a pre-functionalised commercially available resin or may be added to the substrate by subsequent functionalisation or functional group transformation or via the polymerisable reagent which bears the functional group and either reacts with or simply physically interacts with the particle.
  • the substrate is a metal or metal alloy then it may simply be that the reactive functionality of the polymer reacts with and binds to the metal or metal alloy surface.
  • the polymer will also have a reactive functionality, being the same or different to that which binds to the substrate surface, allowing it to crosslink with a plurality of metallic or metallic alloy particles, such as quantum dots, to thereby fix them within the multi-layer polymeric network.
  • the polymer will have, or may be modified to have, a further reactive functionality, including a suitable leaving group, which allows attachment of a desired detection agent.
  • the polymer is not limited to any particular molecular weight range as this may be dependent on the specific application. For example, if the polymer is immobilised in surface sensitive sensors a small molecular weight polymer may be preferred whereas, for particle substrates, a longer molecular weight polymer might be beneficial to extend over the surface roughness.
  • the polymer is PDEA-dextran functional ised with a reactive group, for example 2-(pyridinyldithio)ethyl carbomoyl dextran (PDEC- dextran).
  • a reactive group for example 2-(pyridinyldithio)ethyl carbomoyl dextran (PDEC- dextran).
  • Sulfhydryl groups on the scaffold surface of the loaded substrate, with entrained polymerised MPTS react with the available (2-pyridinyl)disulfanyl group of PDEC-dextran to form a corresponding disulphide bond with the subsequent formation of a 2-mercaptopyridine leaving group.
  • the reactive functionality of the polymer suitable for binding with the scaffold surface, the metal or metal alloy surface and the detection agent comprises a disulfide linkage
  • the leaving group forming part of this functionality is not limited to a 2-pyridinyl moiety.
  • the polymer reactive functionality comprises — S-S-R wherein R is selected from aryl, heteroaryl and C-1-C12 alkyl, each of which may be substituted preferably with electron withdrawing groups.
  • R is heteroaryl and aryl is may be selected from a C5-C7 ring, preferably a C6 ring.
  • R when R is heteroaryl it may be selected from pyrrole, imidazole, pyrazole, pyridine, furan, thiophene, oxazole, thiazole, diazine, oxazine and thiazine.
  • the reactive functionality suitable for binding to the scaffold surface, the metal or metal alloy and the detection agent may be a chalcogen-containing functionality.
  • the reactive functionality comprises two bonded chalcogen atoms each independently selected from S, O, Se and Te.
  • S-S may be preferred.
  • the chalcogen atoms may further have one or two oxygen atoms bonded thereto.
  • the terminal chalcogen atom is attached to an R group such that a good leaving group is formed, for example the reactive functionality may comprise -S-SO 2 -CF 3 .
  • the reactive functionality may comprise: p VJ ⁇ — x wherein each dotted line may represent a bond, each W is independently selected from S, O, Se and Te, O is an oxygen atom which is either doubly bonded to the W or has a further atom attached directly to it and X is an electron-withdrawing atom or group and each W may have further atoms bonded thereto which are not shown.
  • the reactive functionality may comprise:
  • X is an electron-withdrawing atom or group.
  • the electron-withdrawing atom or group may be selected from halogen, C-1-C6 alkylhalide, nitro, nitrile and carbonyl.
  • the binding of the polymer to the scaffold surface preferably happens concurrently with the contacting of the polymer with an additional plurality of metallic or metallic alloy particles, such as a plurality of additional quantum dots, to enable the cross linking reaction to form the three dimensional multi-layer polymeric network rather than a simple monolayer.
  • an additional plurality of metallic or metallic alloy particles such as a plurality of additional quantum dots
  • the plurality of additional metallic or metallic alloy particles may also be selected from the same group as those discussed for incorporation into the polymer matrix of the substrate for example gold, silver, platinum, copper and iron particles as well as quantum dots selected from the group consisting of a lll-V group compound, a ll-VI compound, or a IV group element (which are referred hereon in as a lll-V group quantum dot, a l l-VI quantum dot, or a IV group quantum dot, respectively).
  • the additional metallic or metallic alloy particles are organic quantum dots.
  • the metallic or metallic alloy particles are gold, silver, platinum, copper or iron particles then hydrophilic embodiments are preferred.
  • Q2171 1 MP organic quantum dots
  • the Q2171 1 MP quantum dots comprise a CdSe or CdTe core, a ZnS outer shell to stabilize the core, and an amphiphilic polymer coating around the layer to confer water solubility and to provide surface functional groups for use in physical or chemical interaction with the functional groups of other species.
  • the metallic or metallic alloy particles are quantum dots then they preferably have an average particle size of 0.5 nm to 30 nm, more preferably 1 to 10 nm, and optimally 1 .2 to 8 nm.
  • the plurality of additional metallic or metallic alloy particles within the cross-linked multi-layer polymeric network may have the same functional characteristics as the metallic or metallic alloy particles already entrapped within the loaded substrates, or different functional characteristics depending largely on the specific requirements of the application for which the scaffold is intended.
  • the metallic or metallic alloy particles within the loaded substrate may be magnetic particles and the metallic or metallic alloy particles within the multi-layer polymeric network may be quantum dots.
  • Hybrid fluoro/magnetic particles are already used to advantage in multi-modal imaging, for example MRI combined with fluorescence signalling. It also allows for the option of performing a magnetic enrichment step from a large volume (e.g.
  • the metallic or metallic alloy particles are all quantum dots and those entrapped within the loaded substrates have the same electronic characteristics as the additional quantum dots entrapped within the multilayer polymeric network surrounding the loaded substrates, then the intensity of fluorescence emitted by all of the quantum dots combined will be significantly brighter than in the case where quantum dots are entrapped within the loaded substrates alone.
  • the discussion of the preferred embodiment above has focused on the use of a polymeric matrix containing substrate, such as latex microspheres.
  • the substrate is a gold electrode, or other simple metallic or metallic alloy structure
  • the steps involving swelling in organic solvent, incorporating metallic or metallic alloy particles within the substrate itself and entrapping them therein by use of a polymerisable reagent, such as MPTS are not performed.
  • the metal or metal alloy substrate scaffold surface is simply contacted with the polymer, such as PDEC-dextran, and the metallic or metallic alloy particles which are to be incorporated within the forming multi-layer polymeric network.
  • the polymer reacts with the metal or metal alloy substrate surface to anchor itself there while at the same time a similar reaction with the surface of the metallic or metallic alloy particle effects the crosslinking reaction to form a scaffold comprising a multi-layer polymeric network comprising metallic or metallic alloy particles, such as gold, silver, platinum, copper or iron particles or quantum dots, bound to the metal or metal alloy substrate surface.
  • metallic or metallic alloy particles such as gold, silver, platinum, copper or iron particles or quantum dots
  • the scaffolds of the preferred embodiments of the present invention were biofunctionalized with antibody fragments (mFAbs) according to the protocol described in the Materials and Methods section below and employed in a sandwich- type immunoassay format as described.
  • mFAbs antibody fragments
  • the effect of the multi-layer polymeric network is made clear by the observed greater density of detection agents compared with commercial sensors and the greater sensitivity thereby provided. Such levels of incorporation of detection agent onto a scaffold are not obtainable by use of sensor scaffolds described in the art.
  • the present scaffold allows for immobilisation of a detection agent in greater than 5 times, preferably greater than 10, 20, 30, 40, 50, 60, 70 80 or 90 times the amount which can be incorporated into a scaffold employing the same polymer and detection agent but in a monolayer arrangement.
  • the present scaffold allows for incorporation of detection agent in about 100 times the amount as can be incorporated onto a functionally comparable theoretical monolayer.
  • the present scaffold allows for an at least 5, at least 10, at least 20, at least 30 at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 fold increase in the number of available reactive functionalities suitable for reaction with a detection agent on the multi-layer polymeric network compared with the same polymer in the form of a monolayer covering the same area of scaffold surface.
  • This comparative amount could be measured in a number of ways which would be clear to a person of skill in the art. For example, one useful methodology is described in Saha et al Analytical Chemistry, July 2014. If antibodies or proteins are employed in the biofunctionalization then a BCA assay could provide a quantitative estimation of the number of, for example, IgG molecules present. Further, a functional assay where a fluorescent target is bound to the particles could offer a quantitative estimate of the number of (active) detection agents on the scaffolds.
  • the sensor resulting from binding of the detection agent to the scaffold of the second aspect may, in one embodiment, be provided in the form of an ELISA kit for performing immunoassays, and comprises a plurality of scaffolds prepared according to the embodiments described above in, for example, a Nunc Maxisorp polystyrene multi-well plate 210 (44-2404-21 , as supplied by Nunc).
  • the scaffolds may be modified with a plurality of detection agents using suitable coupling protocols according to the functional groups on the scaffold.
  • the immobilised PDEC-dextran presents a plurality of available (2-pyridinyl)disulfanyl groups at the surface.
  • a detection agent such as an antibody fragment (imFAb), comprising a sulfhydryl group at the free cysteine at the hinge region of the antibody that is capable of reacting with the available (2-pyridinyl)disulfanyl group may be used to form a disulphide linkage and the subsequent formation of a 2-mercaptopyridine leaving group to result in biofunctionalized scaffolds.
  • imFAb an antibody fragment
  • the multi-well plate may be incubated overnight with 100 L/well of mFAb (5 g/mL in carbonate buffer 50 mM, pH 8.5), cross-reactive against all four dengue serotypes.
  • the wells of the resulting biofunctionalized multi-well plate may then be washed with PBSP buffer and subsequently blocked with KPL buffer (KPL50-82-01 ).
  • the biofunctionalized multi-well plate may then be incubated with a test sample comprising a target molecule in the form of NS1 protein, serially diluted in human serum (or PBSP buffer).
  • the NS1 protein is capable of forming a conjugate with the immobilized mFAbs on the surface of the biofunctionalized multi-well plate such that the NS1 protein becomes immobilized on the surface of the biofunctionalized multi-well plate (denoted herein as the bioconjugated multi-well plate).
  • the biofunctionalized scaffolds may then be added to the wells of the bioconjugated multi-well plate and incubated for 1 hour to allow for the immobilized mFAbs on the surface of the biofunctionalized scaffolds to recognise and ligate with the NS1 proteins immobilized on the surface of the bioconjugated multi-well plate.
  • the unbound biofunctionalized scaffolds may be removed via a washing step with PBSP buffer and the fluorescence intensity of the remaining biofunctionalized scaffolds bound to the surface of the bioconjugated multi-well plate can be read with a Perkin Elmer Envision 2104-0010 plate reader to determine the presence and/or concentration of the NS1 protein in the test sample.
  • Step 1 incorporation of QDs by swelling.
  • a 100 ⁇ _ solution of amino-functionalised latex particles (Polybead amino microspheres, 200 nm or 500 nm) was washed twice with butanol by centrifugation (2 minutes at 13k rcf). The washed particles were then suspended in a mixture of toluene-butanol 5:95 and placed in a sonic bath for 60s. A solution of 500 pmol of PL- QD-O-590 quantum dots in toluene-butanol 5:95 was added to the latex particle suspension and then incubated for 30 minutes under gentle shaking in darkness.
  • the fluorescence intensity of a 100 ⁇ _ of supernatant collected at the first wash step is compared with the fluorescence intensity of the solution of the PL-QD- O-590 quantum dots provided at input and a solution of the loaded particles as reference samples to determine the number of PL-QD-O-590 quantum dots incorporated within the loaded particles.
  • a standard curve is obtained by serially diluting the supernatant from the first washing step in 5:95 toluene-butanol.
  • the concentration of quantum dots remaining within the supernatant from the first washing step can be determined using the linear fit inset of Fig. 1 (i), and from this one can estimate the incorporation efficiency of quantum dots within the loaded substrate.
  • a highly efficient incorporation of quantum dots within the loaded substrate is achieved, with a yield higher than 99%.
  • Fig. 2(i) and 2(H) shows the raw data from the Ellman's assay obtained for five different batches of loaded substrate. By comparing with the reference curve, it was estimated that each loaded substrate comprised (5.7 ⁇ 2.2) x10 7 sulfhydryl groups at the surface of the loaded substrate.
  • Step 2 Generation of polymer layer.
  • PDEC-dextran 0.1 mg
  • a 20 pmol solution of Q2171 1 MP quantum dots were added to the loaded substrates obtained at step 1 .
  • the resulting multi-layer polymeric network comprising the additional quantum dots (Q2171 1 MP) was formed and the scaffold was washed in absolute ethanol and resuspended in PBSP buffer (PBS, pH 7.4, 0.1 % Pluronic F127).
  • the quantification of the density of additional quantum dots (Q2171 1 MP) entrapped within the multi-layer polymeric network of the scaffold was carried out following the same protocol as for the incorporation of quantum dots into the substrate, as described in step 1 .
  • the fluorescence intensity of 100 ⁇ _ of supernatant collected at the first wash step is compared with the fluorescence intensity of a solution of the loaded substrates (comprising PL-QD-O-590 quantum dots entrapped therewithin) provided at input and a solution of the scaffold to determine the number of additional quantum dots (Q2171 1 MP) incorporated within the multi-layer polymeric network of the scaffold.
  • a reference curve was obtained by serially diluting the supernatant from the first washing step in 5:95 toluene-butanol.
  • the concentration of the supernatant from the first washing step was determined using the linear fit of Fig. 3(i), and from this one can estimate the number of additional quantum dots (Q21 71 1 MP) remaining in the supernatant after the first washing step.
  • Reference scaffold 0.1 mL of Life-Technologies (F881 1 ) FluoSpheres® carboxylate-modified latex particles were washed into 50mM MES buffer (pH 6 with 0.01 % Tween20). 1.5 mg of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, Sigma Aldrich 39391 ) and 1 .5mg of sulfo-NHS (N-hydroxysulfosuccinimide, Thermo Fisher 24510) were dissolved in deionized water and added right away to the carboxylate-modified latex particles.
  • EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, Sigma Aldrich 39391
  • sulfo-NHS N-hydroxysulfosuccinimide, Thermo Fisher 24510
  • modified latex particles were rapidly washed twice in PBS buffer, 0.1 % Pluronic F127 and mFAb were added and incubated for 2h at room temperature. The reaction was quenched with large excess of ethanolamine. The resulting biofunctionalized latex particles were resuspended in PBSP buffer.
  • Second scaffold the loaded substrates were prepared according to the procedure outlined in Step 1 above, were incubated with PDEC-dextran in the presence of the additional quantum dots (Q21711 MP) within the multi-layer polymeric network according to the procedure outlined in Step 2 above.
  • the BCA assay estimates obtained for the reference scaffold (0.7ug), the monolayer scaffold (3ug) and the multilayer scaffold of the invention (18ug) correspond respectively to a 1.5%, 6% and 36% coupling efficiency between the immobilized mFAbs and IgG.
  • the EDC/NHS step corresponds to a theoretical monolayer, assuming spherical particles with no surface roughness. Since the polymer in this experiment cannot crosslink with quantum dots to form a multilayer polymeric network, only a monolayer is formed by reacting only with the thiols on the surface.
  • Fig. 4(H) presents a dose-response curve obtained with the multi-layer polymeric network containing scaffolds of the invention (labelled Our approach').
  • F881 1 is a functionalised commercial particle. In both curves seen in FIG. 4(iii) the antibody pair used is the same and the curves clearly demonstrate the superior sensitivity and lower limit of detection provided for by the scaffolds of the invention compared to commercial approaches.
  • a batch of multi-layer scaffolds of the present invention was prepared as outlined above and stored at 4°C in darkness. The fluorescence intensity of 10 fmol of the multilayer scaffolds was periodically recorded under optimized readout settings.
  • Two reference samples were also prepared according to protocols adapted from literature methods (Wang, D.S. , et al. , Superparamagnetic Fe203 Beads- CdSe/ZnS quantum dots core-shell nanocomposite particles for cell separation. Nano Letters, 2004. 4(3): p. 409-413; and Koktysh, D., V. Bright, and W. Pham, Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging. Nanotechnology, 201 1 . 22(27).).
  • Amide bond batch 0.1 mL of Polybead Amino Microspheres 07763-5 is buffer exchanged to 50mM MES pH 6, 0.1 % Pluronic F127 and 0.1 mL of Crystalplex -COOH quantum dots (TriliteTM Green, Emission 525 nm) were added. 1 .5mg EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, Sigma Aldrich 39391 ) dissolved in deionized water was added to the mixture and incubated for 2h at room temperature. Unbound quantum dots were removed with three washing steps.
  • EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, Sigma Aldrich 39391
  • Sulfhvdryl/metal batch Polybead 09836-15, functionalized with -COOH groups were surface modified to introduce sulfhydryl groups by means of standard carbodiimide chemistry. The successful modification was monitored by means of an Ellman's assay, following the same protocol as outlined above. A very high density of sulfhydryl groups of 0.5-1 SH/nm 2 was found. The modified particles were suspended in 1 : 1 mixture of toluene/ethanol, to ensure solubility of both the quantum dots and the latex particles. Organic quantum dots (Life Technologies, 545 nm) were added and incubated for 1.5h under gentle shaking. Unbound quantum dots were removed by several washing steps.
  • the multilayer scaffold demonstrates a markedly improved stability over time compared with the stabilities of the reference samples.
  • the multilayer scaffold also demonstrates a superior brightness corresponding to a 100-fold higher density of detection agents immobilized on the surface of the multilayer scaffold as a result of the thickness of the multi-layer polymeric network formed around the loaded substrate. The combination of these advantages results in a 4-fold higher signal and superior sensitivity on a model dengue assay.
  • the magnetic labelling may be accompanied by fluorescent labelling of the scaffold, as described below.
  • polystyrene particles are doped with nanometer-sized ferromagnetic grains and capped with a hydrophilic polymer that enables immobilization of reporter probes and the fluorescent agent.
  • Composite particles were prepared and tested on a model assay to detect the exemplar cardiac biomarker myoglobin, clinically relevant for detection of myocardial infarction.
  • HY_QD has a multi-layer polymer network formed from quantum dots and PDEC-Dextran
  • HY_FP scaffold has a multi-layer polymer network formed from gold nanoparticles and PDEC-Dextran
  • HY_HRP has a multi-layer polymer network formed from gold nanoparticles and PDEC-Dextran with horse radish peroxidase (HRP) enzyme additionally anchored thereto.
  • HRP horse radish peroxidase
  • Fe30 4 were added to separate portions of the microsphere particles to achieve different amounts of incorporated magnetic material in separate batches, and hence a range of magnetic susceptibilities for the scaffolds, and incubated for 1 hour.
  • a catalytic amount of gold nanoparticles (Sigma product# 741981 ; Gold nanoparticles; 40 nm diameter, OD 1 , stabilized suspension in citrate buffer) were added to the microsphere particles. The mixture was incubated for 30 minutes. B-mercaptopropyltrimetoxysaline (MPTS) was added to the mixture and incubated for 20 minutes before adding traces of water.
  • the microsphere particle substrates with incorporated magnetic and gold nanoparticles were finally washed three times with mixture of ethanol-isopropylglycol (1 :1 ).
  • HY QD particles The doped substrates, formed as just described, were divided into three different batches.
  • 10 pmol of quantum dots product P1191580; semiconductor CdSe core and ZnS shell
  • polymer PDEC-Dextran 10 pmol of quantum dots (product P1191580; semiconductor CdSe core and ZnS shell) were added together with polymer PDEC-Dextran.
  • Complete encapsulation of the particles by the polymer to form a multi-layer polymer network was achieved with 4 hours of incubation.
  • the substrate microsphere particles were washed three times with a mixture of ethanol-isopropylglycol (1 : 1 ) and finally resuspended in PBSP buffer (10mM PBS pH 7.4, 0.1 % Pluronic F127).
  • HY FP particles The same approach for the formation of HY_QD particles was followed here, however the QDs were replaced with 10 pmol of nanogold particles to anchor the polymer and form the multi-layer polymer network. Fluorescent probes were then attached to these substrate microsphere particles using a bio-orthogonal copper free click chemistry approach. 5 ⁇ Thiol-PEG-Azido linkers (100 mg/ml) were added to the substrate batches of 20 ⁇ and incubated for 1 hour at ambient temperature. The particles were washed three times with PBSP and resuspended with 20 ⁇ of PBSP. The amount of the azide linker was estimated as follows.
  • Fluorescent probe synthesis protocol DBCO-PEG4-Amine (15 mg, 2.86 x 10-5 mol), PyBOP (16.4 mg, 3.15 x 10-5 mol) and DMACA acid (7.8 mg, 3.15 x 10-5 mol) in DMF (5 ml_) were stirred until dissolve. DIPEA (40 ⁇ _, 2.29 x 10-4 mol) was added slowly to the reaction mixture. The reaction was stirred at room temperature 2 h, followed by removal of solvent under reduced pressure.
  • HRP and antibodies were buffer exchanged to PBS from the original storage buffer using a Zeba spin desalting column (MWCO 7KDa, ThermoFisher, 89882) and following the manufacturer's instructions. Summarizing the protocol, the storage buffer of the column was removed by spinning the column at 1500 RPM for 1 min. 300 ⁇ of PBS was added and the column and buffer removed by spinning. Finally, protein (up to 130 ⁇ ) was added carefully and the column was spun at 1500 RPM for 2 mins. The flow through was collected and the concentration of protein was determined using a ThermoScientific Nanodrop at 280 nm.
  • the unreacted linkers were removed using zeba spin column (MWCO 7KDa, ThermoFisher, 89882).
  • the thiolated antibodies were reacted with the HY_QD, HY_FP and HY_HRP scaffolds, to form the corresponding sensors, for 1 hour at room temperature.
  • the sensors were washed three times with PBSP and finally resuspended with 20 ⁇ of PBSP.
  • Results The number of layers or density of the multi-layer polymer network increases with an increase in the amount of Quantum Dots/gold nanoparticles (or other metallic or metallic alloy particles) which are made available when forming said layers thereby indicating a progressive layer formation. This is important as it provides for an unprecedented level of control over the thickness of the multi-layer polymer network formed on the surface of the particles, and hence the number of fluorescent particles, reporter agents, detection agent etc.
  • Fig. 6 a) - c) demonstrates this increase in the number of layers or density of the multi-layer polymer network by indicating the observed increase in fluorescence of the particle for batches the HY_QDs and HY_FP scaffolds and an increase in HRP activity for the HY HRP scaffold.
  • WGA and ConA were conjugated to magnetic substrate particles of different sizes (170 and 500 nm) and used for the capture of bacterial cells.
  • Lectin coupling was performed with different particle based chemistries, assessed and quantified to select the highest conjugation efficiency. Two chemistries had been tested for determination of lectin coupling efficiency to the surface of the substrate particles: direct coupling to the substrate particle surface or anchoring onto a polymeric hydrogel.
  • the polymeric hydrogel is a functional dextran polymer, synthesized in-house, in which dextran was modified to introduce other functional groups such PDEC (PDEC-dextran) or PDEC and carboxylic acid (CMD-dextran).
  • PDEC-dextran PDEC-dextran
  • CMD-dextran carboxylic acid
  • WGA was attached to carboxylated magnetic substrate particles using standard carbodiimide chemistry.
  • the polymer approach chemistry involved the conjugation of PDEC-dextran polymer (synthesized according to the procedure of Li, X., C. Abell, and M.A. Cooper, Single- step biocompatible coating for sulfhydryl coupling of receptors using 2- (pyridinyldithio)ethylcarbamoyl dextran.
  • Protocol 1 for substrate particle functionalization using CMD-dextran polymer Carboxylated magnetic particles (50 mg/mL) and 0.2 ⁇ FluoSpheres carboxylate-modified Microspheres (20 mg/mL) (Life Technology) were modified and passivated with CMD-PDEC-dextran polymer (Synthesized according to Li and Cooper paper discussed above). Briefly, an equal volume of cysteamine (35 mg/mL) was added to 500 iL of carboxylated nanoparticles then 6.25 mg of carbodiimide EDC was added in cold MES buffer (pH 5.0) and left for incubation for 2 hours at room temperature.
  • washed particles were suspended in CHES buffer pH 9.0 and add 8.5 pL CMD-PDEC dextran (13 mg/mL) in CHES buffer pH 9.0 as well as 10 ⁇ of Ag nanoparticles (10 nm) (Sigma Aldrich) where the Ag nanoparticles were added to build the multi-layer polymeric network.
  • Protocol 2 for capturing bacterial cells from biological samples All bacterial strains were cultured on LB broth at 37 °C and diluted to the required concentrations when their corresponding ⁇ . ⁇ is about 0.5, which corresponds to 10 9 cfu/mL. All biological samples (platelets, plasma, and blood (Australian Red Cross Blood Service (ARCBS)) were sterile and spiked with bacterial strains (Gram- negative and Gram-positive) at different concentrations and processed for magnetic capturing. Bacterial cell capturing was done on 1 mL scale of spiked samples where 3 ⁇ of WGA-MPs (500 nm) or 1 ⁇ of WGA-MPs (170 nm) were added and incubated for 1 hour at 37°C with rotation.
  • All biological samples platelets, plasma, and blood (Australian Red Cross Blood Service (ARCBS)
  • ARCBS Australian Red Cross Blood Service
  • Bacterial cell capturing was done on 1 mL scale of spiked samples where 3 ⁇ of WGA-MPs (500
  • Protocol 3 for quantification of capturing efficiency using culture methods WGA-MPs were magnetically washed with sterile phosphate buffered saline (0.1 % pluronic) (PBSP) 4 times. Then, WGA-MPs were resuspended in 150 ⁇ _ and cultured on LB agar and incubated for 24 hr at 37 °C. Capturing efficiency was calculated based on the number of counted colonies of the output plates in comparison to the input plates.
  • PBSP sterile phosphate buffered saline
  • Fig. 1 1 demonstrates that the polymer (PDEC-dextran) passivation on the magnetic substrate particles (MPs) decreased non-specific binding compared to that seen with standard protein (HSA) passivation.
  • Fluorescent bacterial detection in platelets Fluorescent bacterial detection in platelets.
  • Vancomycin MCC_174 was functionalized on 0.2 ⁇ blue fluorescent (365/415) particles. Fluorescent blue beads were chosen to allow assessment of vancomycin coupling using Fam-KAA, which has overlapping and interfering fluorescence emission with Y/G beads. The surface of the particles was passivated with the polymer (CMD-dextran) based passivation method, through carbodiimide bond (EDC) with cysteamine, before coupling to the vancomycin derivative with another carbodiimide bond (EDC) as to reduce the non-specific binding. The results showed significant improvement of the detection approach with a detection limit of approximately 10 cfu/mL (confirmed by culture) of S. aureus in platelets (results shown in Fig. 12). The extent of the error bars were due to variability between the count of bacteria and manual washing which can be significantly improved by the use of robotics and would therefore lower the calculated limit of detection (LoD).
  • CMD-dextran polymer
  • EDC carbodi
  • Figure 12 therefore exemplifies the results of an assay format where magnetic and fluorescent sensors of the invention simultaneously bind a target analyte (bacteria).
  • Magnetic (due to the inclusion of the magnetic particles in the substrate) sensors are able to be spiked directly into undiluted biological fluids and selectively bind onto the bacterial membrane.
  • the PDEC dextran assists in passivating the surface to reduce non-specific interactions while at the same time enabling immobilization of a very high density of capture probes, essential to achieve rapid capture from large sample volumes.
  • the magnetically sequestered bacteria are then brought into contact with a fluorescent nanoparticle to facilitate fluorescent detection.
  • Enzyme linked immunosorbent assay has been extensively used to successfully detect antigens although it is time consuming and requires several washing steps.
  • a proof of concept assay has been developed that is based on the ELISA sandwich approach.
  • the assay kinetics are somewhat slow due to the Kon of the dengue antibodies (10 4 M “1 S " 1 ), but this will be enhanced by selecting other antibodies.
  • metal nanoparticles can be used to quench the fluorescence signal of quantum dots because of their optical surface plasmon resonance, which leads to the use of that fluorescence energy transfer between quantum dots and metal nanoparticles in diagnostic assays. That energy is called Forster resonance energy transfer (FRET) which is distance dependent.
  • FRET Forster resonance energy transfer
  • metal nanoparticles and quantum dots to detect an antigen as a proof of concept. Since the sandwich formed is through Fabs or antibodies target recognition, the contribution of fluorescence absorption in the detection signal will be investigated.
  • Wash free biomarker assay Amine modified polystyrene beads (200 nm) were doped with fluorescent quantum dots (QDs) as already described in the above experimental, and passivated by PDEC-dextran to form the multi-layer polymeric network as well as reducing the non-specific signal.
  • QDs fluorescent quantum dots
  • another batch of polystyrene beads was loaded internally with silver nanoparticles (AgNPs) or gold nanoparticles (AuNPs), after being modified with PDEC-dextran to form the multilayer polymeric network with the help of nanoparticles and passivate the substrate particles at the same time (protocol 4 below).
  • This metal nanoparticle batch was used to absorb or quench the emitted fluorescence signal of the QDs. Both batches described above were functional ized with either Anti-Dengue Fabs or antibodies. In the presence of specific dengue antigen, a sandwich of the two different batches of substrate polystyrene beads was formed and this resulted in a decrease in the fluorescence signal of the QDs upon excitation. The assay thus developed is wash free and the positive signal is recognised as a decrease in the fluorescence signal in comparison to the negative control (no antigen).
  • the concentration of the metal nanoparticles is directly proportional to the QDs fluorescence absorbed.
  • the use of more metal nanoparticles beads changed the assay kinetics and allowed more binding events to occur resulting in a greater absorbed fluorescence signal for a given incubation time.
  • Protocol 4 for preparation of fluorescence acceptor particles To prepare 200 ⁇ , 40 ⁇ Polybead amine microspheres (20 mg/mL), 0.20 Mm (Polysciences) were washed by 1 : 1 isopropyl glycol : methanol (0.1 % pluronic F127) (S) and reacted with Sulfo-SMCC for 1 hour at room temperature. After which the particles were washed and suspended in CHES and sonicated for 5 min. Then, 1 ⁇ of PDEC-dextran (67 mg/mL) and 40 ⁇ AgNPs (10 nm) (Sigma Aldrich) and incubate for 2 hours at room temperature. Particles were washed by centrifugation at maximum speed for 5 min at RT and suspended in CHES. Then, 25 pg/mL of species specific dengue Fabs were added for 20 mg/mL beads.
  • the scaffolds as defined according to the embodiments of the present invention provide a number of advantages in use. They allow, in certain embodiment, the entrapment of highly fluorescent quantum dots or magnetic particles both within the swollen polymeric substrate and within the multi-layer polymeric network attached to the substrate, and immobilize a greater number of detection agents, fluorescent probes, report agents and the like on the surface of the scaffolds due to the thickness/density of the multi- layer polymeric network. This provides for a sensor exhibiting superior brightness, sensitivity and stability as compared to conventional sensors.

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  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Materials Engineering (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Inorganic Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un polymère qui est doté d'une fonctionnalité pouvant réagir avec des particules métalliques ou d'alliage métallique, utilisé pour former un échafaudage pour un capteur par liaison du polymère à une surface de substrat et par réticulation du polymère avec une pluralité de particules métalliques ou d'alliage métallique pour générer un réseau polymère multicouche en trois dimensions présentant les particules liées en son sein. Le polymère permettra la liaison subséquente d'un agent de détection, tel qu'un anticorps ou un fragment d'anticorps, pour former le capteur.
PCT/AU2015/050564 2014-09-19 2015-09-21 Échafaudage de capteur WO2016041022A1 (fr)

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AU2014903750A AU2014903750A0 (en) 2014-09-19 A scaffold and a method for constructing a scaffold for a sensor for detecting a target molecule in a sample
AU2014903750 2014-09-19

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WO2016041022A1 true WO2016041022A1 (fr) 2016-03-24

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WO2018043687A3 (fr) * 2016-08-31 2018-04-26 積水化学工業株式会社 Particules fluorescentes pour agent de diagnostic et réactif de dosage immunologique l'employant
WO2018102890A1 (fr) * 2016-12-09 2018-06-14 The University Of Queensland Constructions de visualisation
WO2018102889A1 (fr) * 2016-12-09 2018-06-14 The University Of Queensland Produits de synthèse comportant un antibiotique glycopeptidique
CN110312518A (zh) * 2016-12-09 2019-10-08 昆士兰大学 糖蛋白抗生物素构建体
CN110823874A (zh) * 2018-08-13 2020-02-21 博阳生物科技（上海）有限公司 一种均相化学发光检测试剂盒及其应用
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