WO2011045394A1 - Biomolecular labels, involving nanoparticles functionalised with dendrimers - Google Patents

Biomolecular labels, involving nanoparticles functionalised with dendrimers Download PDF

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Publication number
WO2011045394A1
WO2011045394A1 PCT/EP2010/065469 EP2010065469W WO2011045394A1 WO 2011045394 A1 WO2011045394 A1 WO 2011045394A1 EP 2010065469 W EP2010065469 W EP 2010065469W WO 2011045394 A1 WO2011045394 A1 WO 2011045394A1
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Prior art keywords
nanoparticle
dendrimer
label
target analyte
bel
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PCT/EP2010/065469
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French (fr)
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Vladimir Gubala
Xavier Le Guevel
Robert Nooney
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Dublin City University
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Publication of WO2011045394A1 publication Critical patent/WO2011045394A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0409—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is not a halogenated organic compound
    • A61K49/0414—Particles, beads, capsules or spheres
    • A61K49/0423—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
    • A61K49/0428—Surface-modified nanoparticles, e.g. immuno-nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
    • A61K49/0447—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is a halogenated organic compound
    • A61K49/0476—Particles, beads, capsules, spheres
    • A61K49/0485—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
    • A61K49/049—Surface-modified nanoparticles, e.g. immune-nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • 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
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

Definitions

  • the invention relates to biomolecule labels and labelling methods that may be used in bioanalytical applications requiring high selectivity and assay precision.
  • the invention relates to biomolecule labels based on nanoparticles, and more particularly, to the use of dendrimer multilinkers in such label, to bioconjugate biomolecule analytes to the detectable nanoparticle without causing nanoparticle aggregation.
  • fluorescent labels are used for a range of applications including immunosorbent assays 1"2 , immunocytochemistry 3"4 , flow cytometry 5"6 and DNA/protein microarray analysis 7"8 . Fluorescence is preferred because it combines high sensitivity with a low limit of detection (LOD) 9"11 .
  • LOD limit of detection
  • Current research in biomedical diagnostics is moving to inexpensive devices, using biochips that require small samples volumes.
  • fluorescent labels with improved physical and chemical properties are required. For example, better photostability and high fluorescence intensity, with a reproducible signal under a variety of chemical and biological conditions.
  • Nanoparticles are useful as labels for antibodies in immunoassay procedures, where an antigen is captured onto a surface then visualised through its reaction with a labelled antibody, because they result in an intense signal. They can however suffer from some disadvantages in comparison with simple molecular labels, most notably effects of particle aggregation and, a related effect, of non-specific binding to the capture surface.
  • dye-doped silica nanoparticles 12"14 stand out as excellent candidates for improved labels, as it is possible to dope silica N Ps with a large number of fluorophores, increasing the total fluorescence of the label significantly 15"16 . Moreover, the fluorophore is protected inside a silica matrix, increasing photostability 18"19 and quantum efficiency 20"21 .
  • Silica N Ps are also non-toxic, chemically inert, and can be prepared in a range of sizes 22 .
  • silica N P with bioreactive groups such as aldehyde, cyano, isothiocyanate, carboxyl, amine, iodoacetamide, malemide, epoxide, thiol, carbodiimide, and many more. Fuctionalisation may be achieved by surface modification via post synthesis grafting onto the silica surface or during the growth phase of the silica nanoparticles but also after by enabling facile bioconjugation 15 ' 22"23 . It has been noted that problematically, bioassay data obtained using the same sil ica N P la bel with the same captu re antibody, but prepared using different bioconjugation protocols can result in inconsistent results.
  • bioreactive groups such as aldehyde, cyano, isothiocyanate, carboxyl, amine, iodoacetamide, malemide, epoxide, thiol, carbodiimide, and many more. Fuctionalisation may be achieved by surface
  • Tan and colleagues 15 proposed a way of improving col loida l sta bil ity of sil ica N Ps through the addition of a negatively charged non-reactive organosilane alongside the bioreactive organosilane. It is also important to maintain a sta ble colloid after addition of a bi-l inker wh ich may be used to conjugate an antibody to the functionalised N P.
  • the functional ity and nu mber of functional groups on the bi-l inker also affects the overall assay performance.
  • labels comprising metal nanoparticles such as silver, gold and magnetic nanoparticles, functionalised with PAMAM dendrimers having with -N H 2 terminal group chemistries.
  • PAMAM dendrimers having with -N H 2 terminal group chemistries.
  • these labels would require the use of another cross-linker (such as glutaraldehyde).
  • US Patent Publication No. 2008/008483 describes decoration of the surface of metal nanoparticles with sequence of polyelectrolyte layers, onto which, generation 5 of -OH and-N H 2 PAMAM dendrimers terminated with N H 2 groups can be immobilized.
  • the surface groups of the dendrimers would have to be modified with fluorophore for this system to be used as a fluorescence label.
  • the attachment of the biomolecule of interest would require the use of additional bilinker which is likely to affect colloid stability.
  • US Patent Publication No. 2009/208580 discloses the use of dendrimer-entrapped gold nanoparticles (DENP) for imaging of cancer cells.
  • the dendrimers utilize -N H 2 , -OH and -OAcetyl terminated dendrimers chemistries.
  • this method can only be used to entrap nanoparticles smaller than 10 nm, since only up to generation 10 are available, the largest of which have a approximately lOnm.
  • antibodies are sized up to around 20 nm. Accordingly, the skilled person would not have any reason to consider the potential utility of these DENP combinations as efficient fluorescent label for antibodies.
  • N H 2 terminal groups which may be further modified with glycidol to form terminal -OH groups and with succinamic acid to form terminal -COOH groups.
  • These dendrimer systems are then used to entrap preorganized silver ions to form clusters within the dendrimer.
  • the material has been show to exhibit low cell toxicity.
  • the dendrimer-entrapped silver nanocomposites are not sensitized with biomolecules. Furthermore, no effect of such action on the colloidal stability is demonstrated.
  • the reported size of the dendrimer-entrapped silver nanocomposites is only 3-7 nm, which casts doubt on their utility in antibody detection.
  • Pan et al. J. Coll. Int. Sci., 2005, Pan Bi-Feng et al.
  • the focal point of the dendron is anchored to the surface of the magnetite nanoparticle, while the surface groups can be grown to higher generations to provide functionality.
  • the result is a nanoparticle system with a positively charged surface arising from the -N H 3 + terminal groups of the dendrons.
  • Immobilization of proteins which is demonstrated using BSA requires a glutaraldehyde linker that usually causes aggregation, as it neutralizes the overall charge of the NP surface.
  • dendrimers as multivalent linkers could be the use of poly-diacetylene- NH 2 vesicles as described by Liao 32 who report the use of 'hollow balls' prepared by polymerization of 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH 2 ) into a larger vesicle.
  • PCDA-NH 2 10,12-pentacosadiynoic acid 2'-aminoethylamide
  • the surface of such a vesicle is terminated by -NH 2 groups and is further modified by small gold particles in order to increase the surface area of gold electrode for protein attachment.
  • a NP labelled biomolecule must retain its specific binding to analyte, while keeping the non-specific adsorption (NSA) on the substrate to minimum. Therefore the linker must facilitate this requirement by being a linker specific for a particular functional group, for example, an amino acid functional group on a biomolecule.
  • biomolecular nanoparticle based label systems which are not prone to aggregation, show good non-specific binding, high selectivity and which can bind and maintain the active form of the protein.
  • bioassays use superior labels comprising multivalent linkers capable of linking the biomolecular detection species to the nanoparticle label in a way that will not result in a reduction in activity, and which not compromise a dispersion of the nanoparticles.
  • the improved label will lead to high assay sensitivity and ultralow LODs for a desired analyte.
  • nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a sta bil izing effect on the a bsolute value of the zeta potential at the nanoparticle surface.
  • the dendrimer may conjugate with one or more intermediate molecules that in turn may conjugate with the target analyte.
  • the sta bilizing effect is relative to the a bsol ute value of the zeta potential, as the zeta potential can be positive or negative depending on the overal l charge present on the nanoparticle. I n one em bodiment, the sta bilizing effect results from an increase in the a bsol ute value of the zeta potential at the nanoparticle su rface.
  • the electrical sta bility of the nanoparticle is adversely affected desta bil ising nanoparticle colloids and increasing the tendency towa rds nanoparticle aggregation .
  • the dendrimer linkers overcome this problem since they typical ly have ionisa ble chemistries, for example, carboxylate or amine chemistries, wh ich can be manipulated to sta bil ize the charge on the nanoparticle.
  • H igh zeta potential wil l confer particle sta bil ity and accordingly, the colloidal dispersion of the nanoparticles wil l resist aggregation. When the zeta potential is low, attraction exceeds repulsion and the dispersion wil l break and the particles aggregate. Because a zeta potential can be positive or negative, an increase in zeta potential within the present invention is an increase in the a bsolute val ue thereof.
  • Dendrimers of the present invention will at least not desta bilize a zeta potential (lower the a bsol ute valve).
  • a zeta potential having an a bsol ute val ue in mV of from a bout 20 and greater is desira ble (a value that is greater than +20 or less than -20 is desira ble).
  • Suita bly the zeta potential has an a bsolute val ue of a bout 25 or greater such as a bout 30 or greater.
  • the positive effect observed on the a bsolute zeta potential by conjugation of the dendrimers of the invention to a nanoparticle surface may also be considered as a non-desta bilizing effect.
  • the fu nctional group provided on the dendrimer may be a carboxylate or an amine functional group (the nanoparticle needs to have a corresponding group on its surface, for example, amine or carboxylate, respectively).
  • the functional group on the dend rimer is a carboxylate group (-COOH ).
  • the fu nctional grou ps provided on the nanoparticle may be phosphate, amine or carboxylate functional groups or com binations thereof.
  • the nature of the groups are such that they complement each other, for example, where the dendrimer comprises an activata ble carboxylate group, the nanoparticle will be provided with a proportion of amine functional groups to allow dendrimer binding to the nanoparticle surface.
  • the dendrimer may suita bly comprise amine groups.
  • the dend rimer may be terminated with carboxylate (-COOH ) chemistries which can be activated by sulfo N-hydroxysuccinimide and a dehydrating agent such as l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, for exa mple.
  • carboxylate -COOH
  • Activation of the dendrimer -COOH groups is desira ble, since the overal l negative zeta potential of the nanoparticle is maintained, al lowing the nanoparticles to retain colloidal sta bil ity and increases the efficiency of the reaction with biomolecular target of interest.
  • Activation of -COOH groups of the dendrimer with sulfo-N HS results in a formation of sulfo-N HS-ester that spontaneously react with amino-groups of the target antibody.
  • Sulfo-N HS-esters advantageously longer lived than N HS-esters, possess a negative charge and so facilitate production of a more stable product of thermodynamic control.
  • the invention provides an improved ultra sensitive la bel having an optimised l in kage between the nanoparticle la bel and the biological ly relevant analyte molecules of interest, while retaining the negative electrostatic charge on the nanoparticle, thus preventing aggregation .
  • the nanoparticle can then be derivatized for detection using methods known to persons skilled in the art.
  • the nanoparticle surface fuctionalisation comprises a first ionisa ble functional group wh ich is inert to a target analyte and a second ion isa ble group functional group for immo bilising at least one dendrimer onto the nanoparticle surface, wherein the first and second ionisa ble functional groups have a different electrostatic charge and the second ionisa ble functional group is present on the nanoparticle surface in an amount sufficient to prevent nanoparticle aggregation in a col loid sol ution.
  • the dendrimer is preassem bled or is partial ly assem bled before it is immo bil ised onto the nanoparticle surface.
  • the nanoparticle surface must be appropriately treated prior to dendrimer attachment.
  • the majority of the surface of the nanoparticles may be provided with, for example, negatively charged phosphate groups which provide a base of negative charge.
  • the phosphate groups are advantageously inert towards proteins and desirably greatly reduce non-specific binding of non target molecules. This leads to labels having high selectivities and allows for very high assay precision.
  • the first ionisa ble function group is present on the nanoparticle surface in a range of from a bout 87% to 93% of the total nanoparticle su rface area.
  • the nanoparticle surface may be provided with reactive amino (-N H 2 ) groups which facilitate attachment of carboxylate (-COOH) terminated dendrimer. More preferably still, the nanoparticle surface may be provided with from about 5% to about 15% amino (N H 2 ) groups for dendrimer attachment. Suitably, the nanoparticle surface is provided with from about 7% to about 13% of amino (N H 2 ) groups onto which the -COOH terminated dendrimer could be attached. Most preferably, the surface is provided with from about 9% to about 11% of amino (-N H 2 ) groups for dendrimer attachment.
  • the amount of surface coverage may be determined by methods known to the skilled person, for example, ninhydrin assay, wherein the nanoparticles are reacted with a known concentration of ninhydrin which binds to the -N H 2 groups on the nanoparticles and causes a quantifiable colour change which can be detected by UV/fluorescent methods and measured against a calibration curve,
  • the nanoparticle surface is functionalised with predominantly a functional group of one charge wherein this group is inert to the target analyte and that a minimum portion of the nanoparticle surface is functionalised with a functional group of an opposite charge, wherein this latter functional group present in minimal quantity is used to tether or immobilize dendrimer to the nanoparticle.
  • a nanoparticle where a negative functional group which is inert to the target predominates on the nanoparticle surface, with a minimum but sufficient amount of a positively charged group is present to bind dendrimer through carboxylate groups on the dendrimers.
  • the advantage of such a system is that the presence of immobilized -COOH terminated dendrimer on the negative nanoshell enhances significantly the zeta potential by increasing and stabilizing the negative charge at the nanoparticle surface.
  • the inert functional groups do not react with the target analyte and so nonspecific binding is very low.
  • the inventors have found that increasing the percentage of the amino group on the surface of the nanoparticle to over about 20% surface coverage results in immediate aggregation of the nanoparticles.
  • the combination of negatively charged surface phosphate groups with a relatively low % of surface coverage with amino groups and less than about 20% amino coverage provide negatively charged nanoparticles which can react with colloid stabilizing negatively charged carboxylate terminated dendrimers to provide labels that allow for very high target specific binding with low non-specific binding.
  • the negatively charged dendrimer enhances and increases the zeta potential of the nanoparticle, increasing the resistance to agglomeration in a colloid solution.
  • a label comprising:
  • nanoparticle surface is functionalised with
  • said dendrimer having at least one activatable functional group for conjugating the target analyte to the functionalised nanoparticle, the dendrimer has a stabilizing effect on the zeta potential at the nanoparticle surface, and
  • first and second ionisable functional groups have a different electrostatic charge and the second ionisable functional group is present on the nanoparticle surface in a amount sufficient to prevent nanoparticle aggregation in a colloidal solution.
  • one of said charged groups being predominant to prevent agglomeration of the nanoparticle while being inert to a target and the other being sufficient to capture the target but not destabilize to cause agglomeration.
  • the first ionisable functional group which is inert to a target analyte predominates on the nanoparticle surface and set a basic charge.
  • the basic charge is negative.
  • the first ionisable functional group is -COOH.
  • the second ionisable functional group is - NH 2 .
  • the labels of the invention advantageously allows for assays with improved label stability, target binding, assay sensitivity, and LOD of the assay compared to the results achieved using alternative linkers such as neutral linkers.
  • the nanoparticle label can easily be prepared while maintaining the integrity of a colloid dispersion of the nanoparticles.
  • the conjugation method of the present invention utilises dendrimers as multivalent linkers and result in a surprising improvement in attachment of biomolecules onto the surface of NPs.
  • the conjugation method disclosed herein advantageously provides stable colloids, as well as increasing the active surface area of the particle.
  • the special dendrimer -COOH activation method using EDC/NHS or sulfoNHS activation allows the label to bind to and retain target protein activity. This means when the label is used in sandwich assays with an antigen for example, the antigen retains its activity towards antibody despite the label binding reaction. This is advantageous since it allows for high sensitivity and low LOD/LOQ in the assay.
  • Use of neutral linker can deactivate protein recognition ability.
  • conjugation of the analyte to the nanoparticle by a dendrimer linker of the invention has the desirable effect of increasing the zeta potential at the nanoparticle surface.
  • This may be the absolute zeta potential at the nanoparticle surface.
  • These may be groups that are binding groups for binding other moieties to the nanoparticle.
  • these may be non-active groups that have been treated to make them active for bonding.
  • the dendrimer may already have, or may already be, functionalised with groups suitable to bind the dendrimer to a nanoparticle and/or target analyte. Groups that allow such binding will be referred to herein as an "activated” or "activatable” group.
  • such activated or activatable groups include carboxylate or amine groups, which may be activated by methods known in the art.
  • the activation generally renders the groups more reactive towards other corresponding groups, for example, amine or carboxylate groups on other moieties.
  • the dendrimer may need to be activated in order to conjugate it to the nanoparticle surface and/or to conjugate the dendrimer to an amino acid on a protein, a cell, antibody, antigen or other suitable analyte.
  • the activated groups on the dendrimer are susceptible to hydrolysis. Hydrolysis can be employed to form charged groups at physiological pH. Therefore, the electrostatic charge on the nanoparticle is increased and colloidal stability is maintained, despite the modification of the surface.
  • the invention is an improvement over prior art nanoparticle labels which require use of linkers, which when bound to the nanoparticle surface, can detrimentally affect the zeta potential of the nanoparticle. For this reason, prior art nanoparticle labels suffer from problems such as nanoparticle aggregation.
  • the invention provides a label comprising a nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a target analyte to the nanoparticle.
  • a dispersion of dendrimer functionalised nanoparticles according to the invention will desirably have a zeta potential which is increased as a result of the conjugation of dendrimer to the nanoparticle surface.
  • An increased zeta potential can offset a greater tendency of nanoparticles that are larger (by virtue of surface modification) to agglomerate.
  • Such a stabilizing effect on the colloidal dispersion of the nanoparticles is desirable since undesirable aggregation or flocculation of the nanoparticles is hindered. This avoids problems that may occur during an assay, for example, particles "dropping out" of solution and too slow a flow rate where lateral flow microfluidic devices are used for analysis.
  • dendrimer linkers having a plurality of reactive groups, improves the probability of binding of the analyte of interest to the nanoparticle. This increases the reaction yield and surface coverage to provide the basis for a surprisingly sensitive analytic technique which can be used to detect unexpectedly low levels of analyte.
  • a method for preparing a label comprising the steps of:
  • the activatable functional group of the dendrimer is a carboxylate.
  • the first ionisable functional group is phosphate
  • the second ionisable functional group is -N H 2 .
  • the third functional group is -COOH.
  • the dendrimer functionalised nanoparticle may be conjugated to a detection molecule, which in turn detects a target analyte.
  • the detection molecule may be highly selective for a corresponding target analyte. By highly selective it is meant that the selectivity for a moiety approaches near specific.
  • the label of the invention comprises a functionalised nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a detection molecule which is highly selective for a further target analyte thereto, wherein the dendrimer is selected to increase the zeta potential of the nanoparticle.
  • the label comprising the detection molecule can then be used to bind a further analyte of interest in a highly selective manner.
  • the detection molecule in this regard may be highly selective for the further analyte of interest.
  • an antibody as a detection molecule (detection antibody) which is (highly) selective for an antigen (capture molecule) which is the target analyte of interest.
  • the detection molecule may be an antigen, for which a particular anti body (capture molecule) is the target analyte.
  • a particular antibody used as a detection molecule may be used to detect a particular cel l expressing a particular antigenic part of a protein.
  • the la bels of the invention are useful tools for biological discovery and biomed ical detection, medical imaging and therapeutic appl ications such as cel l la bel l ing, targeted drug del ivery, targeted gene del ivery, biosensing, cel l separation, cell purification a nd imaging.
  • Dendrimers are monodispersed compounds with a well -defined structure, in which the num ber and functional ity of the dendrimer functional groups can be tailored to the h ighly selective bioconjugation required.
  • Use of multivalent or multifunctional dendrimers as linkers in bioconjugated nanoparticle la bels leads to a higher efficiency in bioconjugation of, for example, ta rget analytes (e.g., antibod ies or antigens), and overall facil itate an improved assay performance with ultra low LO D capa bility and in a highly sensitive and precise manner. Binding occurs more rapidly and so assays are completed in less time.
  • the rate of the bioconjugation reaction will be proportional to the concentration of the reactants.
  • dendrimers provide more reactive groups and therefore a higher surface concentration, than would likely be achieva ble with other monovalent linkers that don't have multipl icity. Rates of bioconjugation of the target analyte to the nanoparticle la bel are improved.
  • the multivalency of the dendrimer increases the probability that analyte will bind to the nanoparticle surface.
  • the invention also relates to a dispersion of labels of the present invention in a suitable medium, desirably an aqueous medium.
  • the dendrimer is a PAMAM dendrimer or a Newkome dend rimer.
  • the hydrophilic dendrimer is a PAMAM dendrimer.
  • PAMAM dendrimers are quite water-soluble. Newkome dendrimers may require sonication to assist in dissolving them in water. In an embodiment where Newkome type dendrimers are used, generation 1 dendrimer are preferred as the full generation Newkome dendrimer presents carboxylate (-COOH) terminal functional groups.
  • the dendrimer is at least one dendrimer selected from the group consisting of generation 0 to generation 10 dendrimers, incl uding half generations of dendrimers. Most preferred are generations that provide carboxylate (-COO H) terminal chemistries.
  • the dendrimer is a generation 0 dendrimer, a generation 0.5 dendrimer, a generation 1 dendrimer, a generation 1.5 dendrimer, a generation 2 dendrimer, a generation 2.5 dendrimer, a generation 3 dendrimer, a generation 3.5 dendrimer, a generation 4 dendrimer, a generation 4.5 dendrimer, a generation 5 dendrimer, a generation 5.5 dend rimer, a generation 6 dendrimer or a generation 6.5 dendrimer. More prefera bly, the generation size of the dendrimer may be from generation 0 to generation 4.5. The most preferred size is generation 4.5.
  • Dendrimers of generation 4.5 or smal ler are more desira ble since, the cost of the higher generations is significant and accordingly they are not ideally suited for use in an inexpensive disposa ble diagnostic device.
  • Generation G4.5 dendrimers typically provide an optimum balance between cost and performance.
  • the dendrimer is of a generation or half generation which is carboxylate (-COOH ) terminated.
  • the dendrimer is a generation 4 dendrimer.
  • the dendrimer is a generation 0 dend rimer.
  • the dendrimer is a generation 2 dendrimer.
  • the dendrimer is a generation 3 dendrimer.
  • the dendrimer is a hydrophil ic or a hydrophobic dendrimer. Desira bly, the dend rimer is a hydrophilic dendrimer, since a num ber of hydrophobic dend rimers (incl uding some starburst dendrimers) are comprised of conjugated aromatic rings. A num ber of these dendrimers may present water sol u bility issues.
  • the dendrimer and the nanoparticle must have compati ble surface chemistries.
  • preferred nanoparticles may comprise non-functional ised surfaces such as silica surfaces.
  • the silica surfaces of the nanoparticles of the invention are typical ly functional ised with groups necessary to facilitate conjugation to the dendrimer.
  • the nanoparticles may be functionalised with amine groups.
  • the dendrimer l in ker should comprise linking functionalities, for example, carboxylate surface chemistries (or other functional ities capa ble of l in king to amine) so that the groups may react together to l ink the dendrimer to the nanoparticle surface.
  • linking functionalities for example, carboxylate surface chemistries (or other functional ities capa ble of l in king to amine) so that the groups may react together to l ink the dendrimer to the nanoparticle surface.
  • Examples of dendrimers having carboxylate surfaces include whole generation, such as generation 0, generation 1, generation 2, etc., or PAMAM type dendrimers of half generation, e.g., generation 0.5, generation 1.5, generation 2.5 etc. It wil l also be appreciated that if the nanoparticle surfaces comprise surface carboxylate grou ps, then dendrimers using amine surface chemistries may be used.
  • the la bel of the invention comprises a non-magnetic, non-metal nanoparticle, such as a sil ica nanoparticle.
  • the nanoparticle may be a silica nanoparticle or a metal oxide nanoparticle.
  • the nanoparticle may be a sil ica particle.
  • Silica is preferred in many appl ications since it is derivatisa ble with bioreactive functional groups, is biocompatible, wh ile being a relatively inert su bsta nce that prevents agglomeration with other nanoparticles in a dispersion .
  • the label of the invention comprises a silica nanoparticle functionalized with at least one dendrimer for conjugating a target analyte to the nanoparticle, so as to allow labelling of the analyte for detection thereof, wherein the dendrimer linker is selected so as to increase the zeta potential of the nanoparticles in a dispersion of the nanoparticles.
  • nanoparticle may be at least one of dye-doped, magnetic, sil ica or metal doped.
  • the nanoparticle is may be a hybrid nanoparticle comprised of a hybrid material and include, noble metal, q uantum dot or magnetite doped silica nanoparticles.
  • the nanoparticle may be a dye-doped nanoparticle which advantageously provides for fluorescence detection.
  • the dye-doped nanoparticle may be fluorescence optimized, for example, for near infrared ( N I R) electromagnetic radiation. This makes the la bel detecta ble by spectrophotometric techniques.
  • the nanoparticle is a dye-doped sil ica nanoparticle.
  • Near-infrared dye doped sil ica N Ps in which the fl uorescence has been optimized are preferred since at near infrared wavelengths there is low background interference from the fluorescence of biological molecules, solvent, and su bstrates.
  • whole blood has a weak a bsorption in the N I R region, thus reducing the need for whole blood filtering for assays using whole blood .
  • N I R l ight can a lso penetrate skin and tissue to several mill imetres ena bl ing fluorescence detection in vivo, for example, in dermatological or in-vivo diagnostic devices.
  • the nanoparticle may have a diameter in the range of a bout 10 nm to a bout 1000 nanometres.
  • the size of the nanoparticle used will depend on the intended appl ication and the size of the target analyte in question. Smal ler nanoparticles may used for assay involving smal l proteins or nucleic acids, whereas larger nanoparticle la bels may be used for appl ication involving cel ls, for example.
  • the silica surface of the nanoparticle may be functionalised with molecules comprising reactive groups for example at least one one of silane, amines, thiols, phosphates, epoxy, carboxyl, cyano, isothiolcyanate, iodoacetamide, azido, NHS-ester groups.
  • the molecule comprises at least one organosilane and desira bly comprises an organo group comprising at least one of thiol, amine or carboxylate, phosphate, epoxide or isothiocynate functional groups.
  • the surface layer further comprises at least one of a generation 0 to a generation 10 dendrimer and half generations thereof and which may be carboxylate or amine terminated depending on the appl ication.
  • Carboxylate dendrimer chemistries are preferred since they are activatable towards biotargets without the need for use of neutral linkers.
  • the nanoparticle surface is modified to comprise desired functional groups.
  • a suitable organosilane may provide reactive groups, for example, amine functionalities on the surface of the nanoparticle which are suitable for binding to dendrimer having activated (or activatable) carboxylate chemistries.
  • silanes it is desirable to provide phosphonate groups having negative charges on the nanoparticle surface; such negatively charged phosphonate groups are necessary to stabilize the nanoparticle.
  • An example of a phosphonate providing organosilane is trihydroxypropylsilanemethylphosphonate.
  • the labels of the invention may comprise nanoparticles having such functionalised silica surfaces, wherein the reactive groups may be selected from the group of activatable biochemical groups consisting of: thiol, amine or carboxylate, phosphate, epoxide or isothiocynate.
  • the reactive groups may be selected from the group of activatable biochemical groups consisting of: thiol, amine or carboxylate, phosphate, epoxide or isothiocynate.
  • a mixture of phosphate and amine groups are provided on the nanoparticle surface in the ranges discussed previously above.
  • the functionalizing group or groups will depend on the dendrimer linker required and/or other molecules of interest that can be added to the nanoparticles for various reasons.
  • the nanoparticle of the invention may be further functionalised with at least one of a protein, a nucleic acid, or a molecular label, a fluorophore, a nanoparticle or other moiety having a suitable group for binding to a functional group on a surface modified nanoparticle, for example, amine or carboxylic acid.
  • the label of the invention may comprise a nanoparticle having a modified silica surface comprising phosphate, amine or carboxylic groups and combinations thereof, and a plurality of dendrimers. Such dendrimers may be all of the same generation or half generation, or may be of different generations, depending on the application.
  • the surface layer may be further modified to comprise at least one of a protein, a nucleic acid, a molecular label, a molecular tag, a fluorophore or a nanoparticle.
  • the modified silica surface layer may have a thickness in the range of about 1 nm to about 100 nanometres.
  • the target analyte may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte highly selective for a further analyte species.
  • a detection molecule highly selective for target analytes can be employed.
  • An example of a detection molecule is a protein, such as an antibody, an antigen, or other moiety having a group suitable for attachment to a dendrimer of the invention, which may be used to selectively bind to a target analyte (for example, antibody, antigen) in an immunoassay.
  • the detection molecule is highly selective for a target analyte selected from the group consisting of: a cell, a pathogen, a protein, a secondary analyte, a molecular label, a molecular tag, and a nucleic acid.
  • secondary analyte it is meant a moiety that may selectively bind to the target analyte.
  • the secondary analyte may then be probed for, or detected by a detection molecule that is highly selective for the secondary analyte. This is useful where a detection molecule highly selective for the target analyte is not available.
  • the target analyte is a protein, for example, green fluorescent protein.
  • the protein may be an antibody, such as a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen.
  • the detection molecule may be a monoclonal antibody, a polyclonal antibody or an antigen.
  • the antibody is a detection antibody which is highly selective for a target analyte.
  • molecular tag is it meant a peptide sequence which is removeably grafted onto a recombinant protein for reasons including facilitating protein detection in assays, protein purification, etc.
  • molecular label it is meant a chemical moiety with a detectable property, for example, a fluorescent or radioactive molecules which can be attached to a nucleic acid as a detectable group.
  • the molecular tag may be biton or streptavdin.
  • the molecular label may be a chemosensor, a fluorescent or a radioactive probe.
  • the fluorescent probe is green fluorescent protein.
  • the nucleic acid is a DNA or an NA.
  • the target analyte, the detection molecule or the secondary analyte may be a protein, including a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen.
  • the label of the invention comprises a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a detection molecule which is highly selective for a corresponding target analyte, wherein the dendrimer increases the zeta potential of the nanoparticle.
  • the detection molecule is a monoclonal antibody, a polyclonal antibody or an antigen.
  • the antibody may be goat antihuman IgG.
  • the antigen may be polyclonal human IgG.ln another aspect of the invention, there is provided a method of preparing a nanoparticle la bel for detecting a target analyte comprising the steps of: (i) activating at least one functional group on a provided nanoparticle to provide at least one activated functional group having enhanced reactivity; and
  • the label may be reactivated before use.
  • the method may further comprise the step of attaching at least one detection molecule to the activated label to form a label.
  • the detection molecule is highly selective for a target analyte.
  • the nanoparticle may be a dye-doped nanoparticle or a magnetic doped nanoparticle. It is preferred that dye-doped nanoparticles are used.
  • the nanoparticle label formed will comprise an activated (or activatable, if quenched after initial preparation) dendrimer linked to a nanoparticle.
  • an activated label is formed.
  • the thus formed activated label may be used immediately in an assay. Alternatively, the activated label may be quenched and stored until required for use.
  • the functional groups on the dendrimer may be activated at a later time, when required for use in an assay or other application.
  • the label of the invention may be used in an immunoassay.
  • the immunoassay is an immunosorbent assay, such as an ELISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.
  • the label of the invention may be used as a tool for biological discovery and/or biomedical detection, in medical imaging and/or therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.
  • the label of the invention may be used in a dermatological or in vivo diagnostic device.
  • a method of detecting a target analyte in a test substance using the label of the invention comprising the steps of:
  • test substance for bound label.
  • the test substance is exposed to the activated label under conditions that allow the activated functional group to bind to the target analyte.
  • the label of the invention may be used in an immunoassay.
  • immunoassay it is meant an assay which measures the concentration of a biochemical substance in a biological liquid, such as serum or urine.
  • assays are generally based on the reaction of an antibody to an antigen.
  • a method of directly detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of:
  • the target analyte may be may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte which is highly selective for a further analyte species.
  • the immunoassay may be an immunosorbent assay, an ELISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.
  • the immunosorbent assay is a plate-based assays for detecting and quantifying biochemical substances, for example, peptides, proteins, antibodies and hormones, etc.
  • the target analyte may then be directly detected by exposing it to a suitable label of the invention, for example a nanoparticle label having dendrimer linked to a primary antibody, which is highly selective for the target analyte.
  • a suitable label of the invention for example a nanoparticle label having dendrimer linked to a primary antibody, which is highly selective for the target analyte.
  • the target analyte may be indirectly detected, by exposing the target analyte to a secondary analyte molecule (for example, a primary antibody which is highly selective for a target antigen) before using a label of the invention comprising a primary antibody as a detection molecule that is highly selective to the secondary analyte molecule (nanoparticle label having dendrimer linked to a detection molecule (for example, primary antibody) which is highly selective for the secondary analyte molecule.
  • a secondary analyte molecule for example, a primary antibody which is highly selective for
  • Immunosorbent assays do not usually use direct detection methods.
  • a detection molecule for example, a secondary antibody
  • a secondary analyte molecule for example, a primary antibody which is highly selective for the target analyte
  • the target analyte is detected indirectly through by probing with the detection molecule for a secondary analyte.
  • indirect detection involves using a primary or secondary antibody that is labelled with an affinity tag such as biotin. Then a secondary (or tertiary) probe, such as streptavidin that is labelled with the nanoparticle label of the invention, can be used to probe for the biotin tag to yield a detectable signal.
  • an affinity tag such as biotin.
  • a secondary (or tertiary) probe such as streptavidin that is labelled with the nanoparticle label of the invention, can be used to probe for the biotin tag to yield a detectable signal.
  • a method of detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of:
  • the target analyte for example, an antigen
  • the support is a microplate or a plurality of polystyrene beads or a gel film or the like.
  • the immunosorbent assay may be performed in 96-well (or 384-well) polystyrene plates, which immobilize antibodies and proteins, including antigen. Using a plate assists in the separation of the bound material from the unbound material during the assay.
  • unbound material may be washed away to facilitate the assay.
  • the target analyte may be a protein, a detection molecule or a secondary analyte which is selective for a further analyte species.
  • the detection molecule and/or the secondary analyte is an antibody.
  • an antibody that recognizes the target analyte is called the "primary antibody”.
  • the primary antibody is linked to a nanoparticle label of the invention, then direct detection of the analyte is possible. This technique is useful for immunohistochemical staining of tissues and cells.
  • the target analyte may then be complexed with the detection molecule (for example, an antibody), which then forms part of the label of the invention.
  • the immunosorbent assay is a sandwich assay.
  • This is desirable since it is one of the most powerful immunosorbent assay formats.
  • This is a type of capture assay which is called a "sandwich" assay because the target analyte to be measured is bound between two primary antibodies, i.e., the capture antibody which is used to immobilize the target analyte onto a support and the label of the invention which comprises a detection antibody which is selective for the target analyte.
  • the sandwich format is preferred since it is a sensitive and robust technique.
  • a method of detecting a target analyte in a sandwich immunosorbent assay using the label of the invention comprising the steps of:
  • This type of method involves an indirect immunosorbent assay procedure in which the analyte is detected, by actual detection of a secondary analyte, which is probed for, by the detection molecule of the label of the invention.
  • the key step of the assay is the immobilization of the target analyte to the support phase (for example, an antigen).
  • immobilization of the target analyte can be accomplished by direct adsorption to the assay plate.
  • the target analyte may be immobilized onto the support phase indirectly through use of a capture molecule (for example, an antibody) that has been attached to the plate before it is exposed to the target analyte.
  • the first step of immobilising the target analyte onto the support may comprise:
  • the method involves directly detects the target analyte through an immunosorbent assay, where the target analyte is bound to a capture molecule which is itself immobilised on the support.
  • the capture molecule may be an antibody or an antigen.
  • the assay methods described herein involve a step of providing a nanoparticle label comprising at least one activatable dendrimer for linking the label to a target analyte, the dendrimer comprising at least one activatable functional group; and activating at least one of the activatable functional groups on the dendrimer to form an activated label.
  • the activated label may then be immediately used in an assay.
  • the methods may be carried out under conditions that allow the activated functional group of the dendrimer on the activated label to bind to the target analyte or to the secondary detection molecule.
  • the target analyte therefore links to the label through the detection molecule or through the secondary analyte as the case may be.
  • the test substance may then be analysed for target analyte bound nanoparticle.
  • the nanoparticle label such as a dye-doped nanoparticle.
  • the target analyte may be protein, a nucleic acid, an antibody or an antigen.
  • the target analyte is a protein, such as GFP.
  • the target analyte is an antigen, such as human IgG.
  • the capture molecule is an antibody, such as goat antihuman IgG.
  • the detection molecule is an antibody, such as goat antihuman IgG.
  • the test substance may be tissue, blood or a tissue or blood derivate.
  • the support is a micro plate or a plurality of polystyrene beads.
  • the support is washed with a buffer, such as N-morpholinoethylsulfonic acid (MES) or Phosphate buffer saline (PBS), polysorbate materials (including polysorbate 20 and 80 such as those sold under the brand name Tween ® ), sodium dodecyl sulphate.
  • MES N-morpholinoethylsulfonic acid
  • PBS Phosphate buffer saline
  • polysorbate materials including polysorbate 20 and 80 such as those sold under the brand name Tween ®
  • sodium dodecyl sulphate sodium dodecyl sulphate.
  • the test substance may be tissue, blood, for example, whole blood or a blood or tissue derivate.
  • the test su bstance sample may be worked up (cleaned up) before assay.
  • the activating step necessary to form an activated label involves treating the nanoparticle label (comprising activata ble dendrimer, or activatable carboxylate groups on the nanoparticle, if provide in this order) with N-hydroxysuccinimide (N HS) or sulfo N-hydroxysuccinimide, and a dehydrating agent.
  • N HS N-hydroxysuccinimide
  • Sulfo N-hydroxysuccinimide and a dehydrating agent are the preferred activating reagents, since use of sulfo N-hydroxysuccinimide provides a more stable solution of the nanoparticle conjugates.
  • the dehydrating agent may be l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC).
  • EDC l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
  • This activating system is capable of activating carboxylate groups on the dendrimer to enhance their reactivity towards amine groups on the nanoparticle surface, (or vice versa, where the nanoparticle comprises activatable carboxylic acid groups and the dendrimer comprises amine surface functionalities). It will be appreciated that other activating system may be suitably used to activate other types of functional groups.
  • the nanoparticle label may be activated by treating the nanoparticle label with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC).
  • NHS N-hydroxysuccinimide
  • EDC dehydrating agent l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride
  • the dendrimer carboxylate functional groups are made more reactive towards amines groups.
  • Amine groups can be found on the nanoparticle surface. Amine groups can also be found on suitable target analyte or detection molecules.
  • the reaction may take place in a MES buffer.
  • the buffer may have a concentration in the range about ImM to about 1M.
  • the pH of the buffer may be in the range of about 3 to about 9.
  • the reaction may proceed for an activation period in the
  • the dendrimer has a carboxylate surface chemistry
  • the ratio of EDC:NHS may be 4:1.
  • the N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide may be present in a two fold molar excess of NHS reagent per carboxylate group (2:1).
  • the label comprises activated dendrimer surface modified nanoparticle comprises semi-stable amine reactive NHS-esters.
  • the dendrimers of the nanoparticle label of the invention may be activated more than one. This is desirable, since the dendrimer label may be pre-prepared and sold in preformed form according to application. When required for use, the dendrimer can be activated in the laboratory.
  • a kit for testing for target analyte comprising:
  • the kit of the invention may further comprise activating reagents. Desira bly, the kit may further comprise buffer and/or wash solutions.
  • Figure 1 shows examples of the monovalent and multivalent cross-linkers used in this work
  • Figure 2 shows a micrograph of dye-doped silica nanoparticles
  • FIG. 3 shows dynamic l ight scattering ( DLS) data showing the amount of nanoparticle aggregation (A) as effect of the corresponding l in ker attached to its surface and ( B) change in zeta potential as a function of the change on the su rface of the N I N P;
  • DLS dynamic l ight scattering
  • FIG. 4 shows normalized fluorescence intensity measured after immobil ization of target analyte Green Fluorescent Protein (G FP) onto the G2 modified N P su rface under various cond itions;
  • G FP Green Fluorescent Protein
  • Figure 5 shows normalized fluorescence intensity measured after immobil ization of target Green Fluorescent Protein on the N P su rface modified with sulfo-SMCC or the G2 dend rimer;
  • Figure 6 shows fluorescence l inked immunosorbent assays for the detection of h lgG using monovalent and bi-l inkers a nd dend rimer multivalent l inkers;
  • Figure 7 shows the total fluorescence signal over background fl uorescence signal, F/Fo combined with coefficient of variance, and LO D results for each l inker in the fluorescence l inked immunosorbent assay for detection of human IgG;
  • Figure 8 shows the rate of change in signal as target concentration changes
  • Figure 9 shows comparison in the surface coverage of N I R-N Ps when using two different types of dendrimers
  • Figure 10 shows binding rate of N I R-N P conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM- N P conjugates in a direct binding assay (right);
  • Figure 11 shows the reaction parameters for the dendrimer-activated nanoparticles relative to those observed for an assay performed using antibody coupled directly to the fluorescent dye;
  • Figure 12 shows a schematic of the surface fuctionalisation of a preferred nanoparticle of the invention, binding to dendrimer multivalent linker and binding to target antibody.
  • Figure 1 schematically illustrates a number of the monovalent and multivalent cross-linkers used in this work.
  • the generation 0 (GO) dendrimer is a tailor made Newkome type dendrimer having seven surface available carboxylate groups.
  • Generation 1 and generation 2 dendrimer are commercially available Newkome type dendrimers obtained from Frontier Scientific.
  • FIG. 2 there is presented a micrograph of dye-doped silica nanoparticles which are approximately 71.16 +/- 3.83 nm in total diameter.
  • the shell thickness is approximately 9.00 +/- 1-80 nm.
  • This layer comprises an area of silica on the surface of the nanoparticle comprising trihydroxypropylsilanemethylphosphonate (for stabilising the nanoparticle) and amino groups for attachment of the dendrimer linker to nanoparticle surface.
  • FIGs 4A and 4B and initially Figure 4A in which there is presented normalized fluorescence intensity measurements taken after immobilization of target analyte Green Fluorescent Protein (GFP) onto the G2 modified N P surface under various conditions.
  • GFP Green Fluorescent Protein
  • Figure 4A it can be seen that by varying the stoichiometric ratio between EDC/N HS for the activation of G2 dendrimer carboxylate groups, the most target protein is bound when the ratio of EDC/N HS is 4:1.
  • Figure 4B shows the effect of varying the activation time of the G2 dendrimer carboxylate groups before reacting with the amino fu nctional ized surface of the N Ps in the first conjugation step.
  • FIG. 6 shows results of fl uorescence l in ked immunosorbent assays for the detection of h lgG using monovalent and bi-l inkers and dendrimer multivalent l inkers.
  • H igher fl uorescent signal are observed when la bel comprising a dend rimer surface modified dye-doped N P is used at the same concentration of target analyte, even at lower concentrations of target human IgG. This clearly indicates that dendrimer l inkers al low a more sensitive assay.
  • FIG 8 shows that rate of change in signal as target concentration changes.
  • the drawing shows a change in the sensitivity of fluorescence for immunosorbent assay with change in concentration of hu man IgG at low concentration . No sensitivity was observed for monovalent l inkers and the organic fluorophore la bel, Cy5 at low concentrations; Signal was detected at low concentration of target when dendrimer surface modified dyedoped N P la bels were used.
  • FIG 10 shows the binding rate of N I R-N P conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM-N P conjugates in a direct binding assay (right).
  • the Figu re shows the reaction kinetics in a direct binding assay when the nanoparticles are sensitized through PAMAM G 1.5 to G4.5. A proper fit was appl ied to the plot, based on which Kon and Koff rates were determined.
  • the 'on' rate, k on c increased with increasing generation num ber of the dend rimer, consistent with the expected increase in the active surface area.
  • N I R l ight can also penetrate skin and tissue to several mill imetres ena bl ing fl uorescence detection in dermatological or in-vivo diagnostic devices.
  • the three bivalent l inkers used are shown in the top half of Figure 1.
  • the structure and mechanism for attachment of the dendrimers is shown in the bottom half of Figure 1.
  • Nanoparticles were characterized using transmission electron microscopy and dynamic Light Scattering.
  • num ber of dendrimers that may be accommodated on the nanoparticle surface wil l depend on the num ber of free amine groups provided on the nanoparticle surface.
  • a nanoparticle of the invention may comprise surface amine groups on in the range of approximately 7 to 13% of the surface, with the remainder of the surface comprising sta bilizing phosphate groups.
  • gl utaraldehyde which may be considered a bi-l inker having two potential l in king sites, binds with one of its aldehyde groups to al l of the free a mine groups present on the nanoparticle surface having 13% amine covered surface, on ly 13% of the surface is availa ble to bind to a target protein.
  • the generation 0 dendrimer binds through one of its seven linking sites to al l of the free amine groups present on the nanoparticle surface having 13%, there are stil l another six linking sites availa ble to bind protein.
  • the num ber of potential protein or target binding sites is su bstantial ly increased by use of a dendrimer l in ker. While it is assumed that the dendrimer binds to the nanoparticle by a single linking -COOH group, it is possible that the dendrimer cou ld bind to the nanoparticle surface by more than one carboxylic acid group.
  • the multivalency of the dendrimer means that it is possi ble that each dendrimer may l ink to multiple amines on the surface of the N P, assuming that the amines are in close to each other.
  • num ber wil l also depend on the size of the protein and wil l increase with increased generation of the dendrimer.
  • the type of target is also an important factor, for example, the num ber will vary depending on the protein, it wil l be different for the whole anti body as for some anti body fragments such as Fa b and ScFv.
  • Figure 12 is a schematic of fuctionalisation of a nanoparticle with the preferred chemistry of the invention, immobilisation of dendrimer on the functionalised nanoparticle surface and use of the thus formed label in a bioassay.
  • the nanoparticle surface may be functionalised using a mixed of derivatized silanes, for example, phosphate and amine derivatized silane. In this particular example, a ratio of 10:1 phosphate compound to amine compound is used.
  • This forms the functionalised nanoparticle surface which preferably comprises in the range 7 - 13% surface coverage of -N H2 groups.
  • Schematic (2) shows the "sensing layer" of dendrimer immobilised onto the functionalised nanoparticle surface and the subsequent attachment to target proteins through activation of the dendrimer terminal -COOH groups with sulfo-N HS/EDC coupling. This type of activation preserves the negative charge on the dendrimer and is crucial for colloidal stability of the resulting nanoparticle-dendrimer label.
  • Triton ® X-100 (trademark Union Carbide), n-hexanol (anhydrous, >99%), cyclohexane (anhydrous 99.5 %), ammonium hydroxide (28 % in H 2 0 > 99.99 %), tetraethylorthosilica (TEOS, 99.99 %), aminopropyltrimethoxysilane (APTMS, 97 %), aminopropyltriethoxysilane (APTES, 99 %), 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (TH PM P, 42 wt % in water), triethylamine (>99 %) absolute ethanol, monobasic sodium phosphate, dibasic sodium phosphate, phosphate buffered saline (PBS, pH 7.4, 0.01 M), Tween ® 20 (trademark Uniqema), glutaraldehyde (25 wt % in water), sodium
  • Dendrimer generation 0 was synthesized in two simple steps starting from cyanuric chloride as starting material and purified on silica gel chromatography column prior the use 43 .
  • Polyclonal Cy5 conjugated goat antihuman IgG (2.5 mg/mL in PBS), polyclonal goat anti human IgG (5 mg / mL in PBS) and polyclonal human IgG (5 mg/mL in PBS) were purchased from Biomeda Corp, California, USA.
  • Black 96 well plates used in the immunoassay were purchased from AGB Scientific Ireland. Deionised water ( ⁇ 18 ⁇ ) was obtained from a Milli-Q system from Millipore Ireland.
  • the dye used in this work is 4,5-Benzo-l'-ethyl-3,3,3',3'-tetramethyl-l-(4- sulfobutyl)indodicarbocyanin-5'-acetic acid N-succinimidyl ester, or more commonly referred to as N I -664-N-succinimidyl ester (purchased from Sigma Aldrich).
  • This dye has a quantum efficiency of 23 %, a molar absorptivity of 187,000 L mol "1 cm “1 and fluorescence excitation and emission wavelengths of 672 nm and 694 nm, respectively, in isopropanol 30 .
  • This dye is usually not used in immunoassays because it is not sol u ble in water.
  • N I -664-N-succinimidyl ester was dissolved in 5 m L anhydrous n-hexanol.
  • 5.021 ⁇ of pure APTES and 3 ⁇ of triethylamine were added.
  • the mixtu re was agitated for 24 hours to ensure conjugation of the N I R dye to the organosilane.
  • the microemulsion was formed by mixing cyclohexane oil phase ( 15 m L), n-hexanol co-solvent (3.256 m L) and Triton ® X-100 surfactant (3.788 g) in 30 m L plastic bottles.
  • microemulsion To form the microemulsion, 0.96 m L of deionised water was added and the solution stirred for five min utes. Fol lowing this, 0.2 m L of TEOS and 0.16m L of N H 4 OH were added to start the growth of the silica N Ps. After thirty minutes, 0.344 m L of the N I R-664-APTES conjugate was added. The reaction was stirred for 24 hrs, after which 0.1 ml TEOS was added with rapid stirring.
  • the organosilane, 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (TH PM P), (42 wt % in water) was added with stirring to prevent aggregation of the nanoparticles 19 .
  • 0.02 m L of bioreactive organosilane, aminopropyltrimethoxysilane, (APTMS) was added to and the solution stirred for a further 24 hou rs.
  • the APTMS has a free primary amine group for crosslinking to biomolecules.
  • the N Ps were separated from the solution with the addition of excess a bsolute ethanol and centrifuged twice with ethanol and once with deionised water. Sonication was used between the washing steps to resuspend the N Ps.
  • the N Ps were dispersed in deionised water, at 2.0 mg / mL and stored in the dark at 4°C.
  • the nanoparticles as described herein comprise surfaces modified with -N H 2 groups.
  • EDC/N HS 1- ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (N HS)
  • GO 4 ⁇ , 7x -COOH
  • M ES buffer 0.5 mL
  • N HS 42 ⁇ , 1.5 equiv. per one -COOH group
  • EDC 168 ⁇ , 6.0 equiv. per one -COOH group
  • the clear liquid phase containing the N HS activated dendrimer was then directly added into the NI R-N P (2mg/mL), while keeping the total volume to 1 mL.
  • a sandwich assay format was used to test the performance of the NPs compared to that of a single dye label.
  • 100 ⁇ of capture antibody polyclonal goat anti human IgG, at 5 ⁇ g / mL was added to each well of a standard 96 well microplate. The plate was then incubated overnight at 4°C. To remove any non-adsorbed antibody the plate was rinsed three times with PBS and three times with PBS/0.05 wt % Tween ® . To prevent non-specific adsorption, 200 ⁇ of 1 wt % BSA in PBS was added to each well and the plate incubated at 37 °C for 1 hour. The rinse cycle was then repeated.
  • the fluorescence signal including standard deviation at each concentration of human IgG was determined from seven replicate experiments.
  • the complete assay was fitted using a standard sigmoidal logistics fit (see below, left hand side) and the low concentrations of human IgG were fitted using Ik afn allometric power function (see below, right hand side).
  • F c is the fluorescence at concentration
  • C F 0 is the background fluorescence
  • F max is the maximum fluorescence
  • P is the power
  • C 0 is the point of inflection.
  • the LOD of human IgG was defined as the concentration corresponding to a fluorescence signal 3 times the standard deviation of the background signal.
  • the sensitivity of the assay (change in fluorescence signal with concentration of human IgG) was determined at a range of concentrations through differentiation of the allometric power function. Previous work had indicated that the sensitivity of an assay is given without relating it to the concentration at which the sensitivity was measured. This can be misleading, since sensitivity does not change linearly with changing concentration.
  • TEM micrographs were obtained using a Hitachi 7000 Transmission Electron Microscope operated at 100 kV. Images were captured digitally using a Megaview 2 CCD camera. Specimens were prepared by dropping aqueous solutions of the N Ps onto a formvar carbon coated copper grid. Fluorescence measurements were performed on a Safire (Tecan) microplate reader. For N I - 664-N-succinimidyl ester-doped N Ps, the excitation and emission wavelengths were set at 672 nm and 700 nm, respectively. Dynamic light scattering (DLS) measurements were performed on a Zetasizer from Malvern instruments to yield values of zeta potential ( ⁇ ) for the N Ps.
  • DLS Dynamic light scattering
  • the monovalent linkers, glutaraldehyde and sulfo-SMCC have destabilizing effect on the N P with the tendency to form aggregates.
  • Sulfo-SMCC shows two different populations, the first transition corresponds to the formation of nanoparticle trimers or tetramers, with an average diameter of 275 nm, and the second corresponds to larger aggregates with polydispersed characteristics.
  • NP modified with the multivalent dendrimers Gl and G2 show only a small increase in particle diameter, increasing the zeta potential from -29.8 to -47.9 and -46.0 for Gl and G2 respectively.
  • the increased colloidal stability of dendrimer coated N Ps is due to the significant increase in the zeta potential on addition of the linker. This is because the N HS ester activated groups on the dendrimer are susceptible to hydrolysis, which leads to the formation of carboxylic groups that are charged at physiological pH. Therefore, the electrostatic charge on the NP is increased and colloidal stability is maintained.
  • GFP Green Fluorescent Protein
  • the bioconjugation of the GFP on the dendrimer-modified surface was performed via a well-known technique for the conjugation of carboxyl (on the dendrimer) to amine groups (on the GFP) in peptides and proteins.
  • the reaction between carboxylic acids and amines to form stable amides is commonly facilitated by an addition of a carbodiimide, such as l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC).
  • EDC l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
  • EDC itself is not particularly efficient in linking because in the first step it forms an unstable O-acylisourea ester that is prone to fast hydrolysis and regeneration of the carboxyl.
  • the carboxylic acids of the dendrimers were therefore activated in the presence of a mixture of N- hydroxysuccinimide (N HS) and a dehydrating agent, EDC.
  • N HS/EDC mixture is commonly used to convert -COOH groups to semi-sta ble amine reactive N HS-esters.
  • the availability and the lifetime of the NHS esters on the dendritic linker will determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labelled.
  • reaction medium - 0.1 M MES buffer, pH 6.0;
  • the optimal value is usually given by the coupling efficiency, concentration of the target molecules and the amount of the non-specific binding and is usually determined experimentally. Therefore, the effect of the concentration of the protein on the reaction yield was investigated.
  • the coupling efficiency of two linkers, sulfo-SMCC and G2 in the bioconjugation reaction using a model protein (GFP) was compared.
  • the reactions were carried out at two different concentrations of the GFP.
  • First reaction was run with the amount of GFP representing 1.5 equivalent of the theoretical value necessary for surface saturation, and second reaction was performed using 3.0 equivalents.
  • Figure 5 shows the effect of the increased concentration of GFP from 1.5 to 3.0 equivalents on the conjugation efficiency.
  • the normalized fluorescence signal for sulfo-SMCC has nearly doubled at the higher GFP concentration (3.0) when compared to the lower GFP concentration (1.5).
  • Fmax is the total nanoparticle signal (from specifically bound N Ps and non specifical ly bou nd N Ps)
  • Fo is the signal from non specifica lly bound N Ps.
  • the normal ized ratio is Fmax divided by Fo. If this ratio is high then this indicates that there is a lot more specifically bound sample than non specifical ly. Th is is indicative of a good assay.
  • the LOD for the dendrimeric NPs compares favorably with published data on the detection of biotinylated hlgG using avidin labelled silica NPs, where an LOD of 1.9 mg / mL was observed 26 .
  • the NPs prepared using dendrimer generation 1 showed a significantly lower LOD and higher sensitivity than the commercial label.
  • the multivalency of the dendrimers is one of the most significant factors behind the increase in the detection sensitivity.
  • dendrimers have a positive effect on NP stability and aggregation. Moreover, they are more reactive with biological materials, whilst maintaining activity and specificity to the analyte of interest. These properties are required for managing cost, in the design and implementation of biomedical devices, where expensive biological materials are necessary.
  • Dendrimers are structurally appealing molecules that have already shown their high potential in the surface science of bioassay devices and this study confirmed that they are emerging as a powerful tool for the labelling of biomolecules in biomedical diagnostics as well.
  • PAMAM polyamidoamine
  • a point-of-care device requires low limit of detection, high sensitivity and ease of fabrication. But it also requires fast (usually within minutes) and precise detection, with a minimal error caused by a personnel manipulation.
  • the use of dendrimers in the conjugation protocols seems to be a robust and reasonable way to prepare labelled antibodies necessary for ELISA type fluorescence assays with short response times and high sensitivity.
  • biomolecular nanoparticle labels of the invention comprising nanoparticles having activatable carboxylate (-COOH) terminated dendrimers offers several distinct advantages over conventional bi- functional linkers and include,
  • the labels of the invention present an opportunity for improvements of sensitivity in immunoassays through the use of dendrimers as antibody coupling agents for sensitisation of fluorescent nanoparticles.
  • Nanoparticles are useful as labels for antibodies in immunoassay procedures. As stated earlier, they can however suffer from some disadvantages including particle aggregation and nonspecific binding to the capture surface. Another issue in the use of nanoparticles as labels is the fraction of the antibody that is coupled to the particle that is in fact active for the reaction with antigen. This fraction can be rather small, which in turn can lead to diminished sensitivity and worsened non-specific binding.
  • dendrimers as the coupling agents suppresses non-specific binding effects to less than the level exhibited by a molecularly-labelled antibody.
  • the net effect is a significantly improved signal/background.
  • the capture reaction of particles onto the capture surface, can be written P + S - PS with forward rate constant k on and reverse rate constant k off .
  • S denotes an active site on the capture surface.
  • k 0 ff will simply be the dissociation rate constant for the antigen-antibody complex.
  • the association rate, k on will be determined by the probability of a reactive collision between a particle and an un-occupied reactive site on the surface. Particles are in a continuous state of collision with the surface, at a rate determined by the diffusion coefficient of the particles and by their concentration.
  • a reactive collision, leading to coupling of the particle to the surface, would occur when a reactive part of the surface of the particle (an antibody, oriented with the binding site exposed) collides with a reactive part of the surface (an antigen, oriented with the epitope exposed).
  • the probability that the collision will be with an active site on the particle surface will simply be the fraction of the particle surface area that is covered by active antibody. As the particle concentration on the surface builds up, then the particles not only occupy sites but also physically block the surface area, on account of their size. So, the probability that the collision will be with an active site on the capture surface will simply be the fraction of the total surface that is unblocked by bound particles, times the fraction of this available capture surface area that is covered by active antigen, times a coverage-dependent factor that expresses the requirement that a particle requires a space whose smallest dimension is at least as large as the particle diameter, in order that the particle can fit into the space.
  • / denotes the bound fluorescence intensity at time, t, with hypothetical value l max if the surface were fully covered with fluorophore
  • c denotes the particle concentration in the solution.
  • the capture rate constant, k on can be written where k D is the diffusion-limit rate constant for particle consumption by the surface, dependent on the particle radius and the viscosity of the medium, e is the reaction efficiency for a reactive collision, q s is the fraction of the capture surface that is active for the capture reaction, in this case dependent on the surface coverage of adsorbed antigen, and q P is the active fraction of the particle surface, in this case dependent on the surface coverage of active antibody attached to the particle.
  • the objective of surface fuctionalisation with dendrimer is to increase q P .
  • the exponential increase of average cluster volume with time in the reaction-limited aggregation of antibody- sensitised particles in the presence of the multi-valent antigen can similarly be described in terms of the probability of capture of antigen onto the surface of a particle or onto a particle at the outside of a cluster times the probability that this site will collide with an active site on another particle or cluster.
  • the rate constant for average cluster volume increase should increase approximately as (q P ) 2 for a given initial concentration of particles and antigen.
  • Figure 10 shows the fit of the bound fluorescence intensity to equation 1, with the addition of a fixed offset that we take to represent a non-specific adsorption of the sensitised particles.
  • I n Figu re 11 we present the reaction parameters for the dend rimer-activated nanoparticles relative to those observed for an assay performed using anti body coupled directly to the fluorescent dye rather than to the nanoparticles loaded with fluorescent dye.
  • the rate constant k k on c+k of f is o btained directly from the fit. Th us the values of k on c scaled relative to one another can simply be obtained from the relative values of the product k(liim,rerl offset)- The similarity of the val ues of (//, m , re /-/o//set) scaled relative to the dye-coupled anti body with those of k on c similarly scaled implies that k off » k on c. The variation of the rate constant, k, is thus that of the off rate, k of f.
  • the other important characteristic of the la bels to consider for use in an assay is the ratio of signal to background : in this case, the ratio of the equil ibrium fl uorescence (offset corrected) to the non-specific offset.
  • offset corrected the ratio of the equil ibrium fl uorescence (offset corrected) to the non-specific offset.
  • Dend rimer GO Intermediate 1 (0.5 g, 1.1 mmol ) and diglycolic anhydride (3 g, 16 mmol ) were placed in a sealed vessel and irradiated in a CEM microwave at 300 W, 120°C for 5 minutes. U pon cool ing, precipitates were formed, the whole mixture was dissolved in water and the pH was adjusted to 4-5. The sol ution was concentrated and the residue was purified on silica gel ch romatography column with MeO H/Ch ⁇ C ⁇ (5:95) as an eluent to yield oily product (440 mg, 35% yield).

Abstract

A label comprising: a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a stabilizing effect on the zeta potential at the nanoparticle surface. Such a label is desirable since the stabilizing effect results from an increase in the zeta potential at the nanoparticle surface. The dendrimer functionalised nanoparticle may be conjugated to a detection molecule which detects a target analyte. Use of the labels in bioassays allow detection of ultralow LODs.

Description

Title:
BIOMOLECULAR LABELS, INVOLVING NANOPARTICLES FUNCTIONALISED WITH DENDRIMERS
Field of the Invention
The invention relates to biomolecule labels and labelling methods that may be used in bioanalytical applications requiring high selectivity and assay precision. In particular, the invention relates to biomolecule labels based on nanoparticles, and more particularly, to the use of dendrimer multilinkers in such label, to bioconjugate biomolecule analytes to the detectable nanoparticle without causing nanoparticle aggregation.
Description of Related Art
In the bioanalysis area, fluorescent labels are used for a range of applications including immunosorbent assays1"2, immunocytochemistry3"4, flow cytometry5"6 and DNA/protein microarray analysis7"8. Fluorescence is preferred because it combines high sensitivity with a low limit of detection (LOD)9"11. Current research in biomedical diagnostics is moving to inexpensive devices, using biochips that require small samples volumes. To meet this new demand fluorescent labels with improved physical and chemical properties are required. For example, better photostability and high fluorescence intensity, with a reproducible signal under a variety of chemical and biological conditions.
Nanoparticles are useful as labels for antibodies in immunoassay procedures, where an antigen is captured onto a surface then visualised through its reaction with a labelled antibody, because they result in an intense signal. They can however suffer from some disadvantages in comparison with simple molecular labels, most notably effects of particle aggregation and, a related effect, of non-specific binding to the capture surface. Another issue in the use of nanoparticles as labels, is the fraction of the antibody that is coupled to the particle, that are in fact active for the reaction with antigen. This fraction can be rather small, which in turn can lead to diminished sensitivity and worsened non-specific binding.
In particular, dye-doped silica nanoparticles12"14 (N P) stand out as excellent candidates for improved labels, as it is possible to dope silica N Ps with a large number of fluorophores, increasing the total fluorescence of the label significantly15"16. Moreover, the fluorophore is protected inside a silica matrix, increasing photostability18"19 and quantum efficiency20"21. Silica N Ps are also non-toxic, chemically inert, and can be prepared in a range of sizes22. It also relatively easy to functionalize silica N P with bioreactive groups such as aldehyde, cyano, isothiocyanate, carboxyl, amine, iodoacetamide, malemide, epoxide, thiol, carbodiimide, and many more. Fuctionalisation may be achieved by surface modification via post synthesis grafting onto the silica surface or during the growth phase of the silica nanoparticles but also after by enabling facile bioconjugation15'22"23. It has been noted that problematically, bioassay data obtained using the same sil ica N P la bel with the same captu re antibody, but prepared using different bioconjugation protocols can result in inconsistent results. For example, previous work involving glutaraldehyde gave inconsistent results. Assays lack precision with large standard deviations between the resu lts. Moreover, each step in the conjugation process can decrease colloidal sta bil ity. Loss of colloidal sta bility can often occur as a result of bioconjugation to a nanoparticle and is a significant stu mbling block in the development of improved sil ica N P la bels.
In 2006, Tan and colleagues15 proposed a way of improving col loida l sta bil ity of sil ica N Ps through the addition of a negatively charged non-reactive organosilane alongside the bioreactive organosilane. It is also important to maintain a sta ble colloid after addition of a bi-l inker wh ich may be used to conjugate an antibody to the functionalised N P.
The functional ity and nu mber of functional groups on the bi-l inker also affects the overall assay performance.
There is a great choice of commercial hetero- or homo-functional l inkers availa ble; however, issues may arise from their effect on N P sta bil ity, aggregation, and additionally, information relating to the solu bil ity and the efficiency of bioconjugation can be lacking. There are many commercial ly availa ble bi-l inkers, for example gl utaraldehyde24, ad ipic acid25, and succinic anhydride22,26. Using glutaraldehyde, the biomolecu le of interest is immobilized directly, without use of activating reagents via Sch iff base formation, however, use of neutral linkers, such as glutaraldehyde negatively effect nanoparticle colloidal sta bil ity. I n many other cases another issue that arises stems from that fact that additional chemicals are required for activation and that several steps may be req uired to conjugate a target to the nanopa rticle. Sta bil ity may be adversely affected since the risk of decrease in the zeta potential is greater.
Previously, a number of groups have focused on the use of labels comprising metal nanoparticles such as silver, gold and magnetic nanoparticles, functionalised with PAMAM dendrimers having with -N H2 terminal group chemistries. In all cases, where a high density attachment of biomolecules onto the dendrimer-functionalized-magnetic/Au/Ag nanoparticles is desired, these labels would require the use of another cross-linker (such as glutaraldehyde).
US Patent Publication No. 2008/008483 describes decoration of the surface of metal nanoparticles with sequence of polyelectrolyte layers, onto which, generation 5 of -OH and-N H2 PAMAM dendrimers terminated with N H2 groups can be immobilized. The surface groups of the dendrimers would have to be modified with fluorophore for this system to be used as a fluorescence label. Furthermore, the attachment of the biomolecule of interest would require the use of additional bilinker which is likely to affect colloid stability. While groups such as Maly27'28 and Shi29,30 have reported a method of using dendrimers as carrier molecules for encapsulation of metal nanoparticles in a dendritic matrix, the size and consequently the active surface area of the gold/silver dendrimer-encapsulated nanocomposites discussed were largely limited by the generation of the dendritic capsule. In both cases, the reasonable size required for attachment of small recognition elements, let alone larger biomolecules such as antibodies and oligonucleotides, required the use of expensive larger dendrimers ranging from generation 5 to 7.
US Patent Publication No. 2009/208580 discloses the use of dendrimer-entrapped gold nanoparticles (DENP) for imaging of cancer cells. The dendrimers utilize -N H2, -OH and -OAcetyl terminated dendrimers chemistries. However, this method can only be used to entrap nanoparticles smaller than 10 nm, since only up to generation 10 are available, the largest of which have a approximately lOnm. Typically, antibodies are sized up to around 20 nm. Accordingly, the skilled person would not have any reason to consider the potential utility of these DENP combinations as efficient fluorescent label for antibodies.
Lesniak et al. (Nanoletters, 2005) have reported generation 5 PAMAM dendrimer having with
N H2 terminal groups which may be further modified with glycidol to form terminal -OH groups and with succinamic acid to form terminal -COOH groups. These dendrimer systems are then used to entrap preorganized silver ions to form clusters within the dendrimer. The material has been show to exhibit low cell toxicity. The dendrimer-entrapped silver nanocomposites are not sensitized with biomolecules. Furthermore, no effect of such action on the colloidal stability is demonstrated. The reported size of the dendrimer-entrapped silver nanocomposites is only 3-7 nm, which casts doubt on their utility in antibody detection.
Pan et al. (J. Coll. Int. Sci., 2005, Pan Bi-Feng et al.) describe a method for functionalizing the surface of magnetic nanoparticles with dendrons, which are synthesized in situ on the surface of the magnetic nanoparticle. The focal point of the dendron is anchored to the surface of the magnetite nanoparticle, while the surface groups can be grown to higher generations to provide functionality. The result is a nanoparticle system with a positively charged surface arising from the -N H3 + terminal groups of the dendrons. Immobilization of proteins which is demonstrated using BSA requires a glutaraldehyde linker that usually causes aggregation, as it neutralizes the overall charge of the NP surface. However, no evidence has been provided to show that the negatively charged BSA is not simply adsorbed on the positively charged surface. The skilled person would find it difficult to envisage that the purported best sample (shown in Figure 2C to aggregate in clusters) could be further used to effectively immobilize proteins such as antibodies.
Gu31 and co-workers attempted to directly grow dendritic structures on the surface of magnetite nanoparticles in an attempt to prevent aggregation. The post-synthetic modification of the magnetite surface involved many synthetic steps including reaction with alkylsilanes (aminopropyltriethoxysilane), methacrylates, ethylenediamine and glutaraldehyde before biomolecule of choice could be immobilized on the surface of the dendrons on the magnetite nanoparticle. While this method is certainly interesting and creative, it is difficult to envision how it could be transferred to larger, industrially scaled production. Furthermore, the dendron modified nanoparticle still requires a cross-linker such as glutaraldehyde to immobilise protein onto the nanoparticle. The effect in the final step of using neutral cross-linker such as glutaraldehyde to immobilize biomolecules on the overall dispersivity of the magnetic nanoparticles was not documented. In the present inventors' experience, multiple synthetic steps and indeed use of glutaraldehyde to link the biomolecule have a negative effect on colloid stability, monodispersity of protein loaded nanoparticles and reduced assay precision.
Other alternatives to dendrimers as multivalent linkers could be the use of poly-diacetylene- NH2 vesicles as described by Liao32 who report the use of 'hollow balls' prepared by polymerization of 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH2) into a larger vesicle. The surface of such a vesicle is terminated by -NH2 groups and is further modified by small gold particles in order to increase the surface area of gold electrode for protein attachment. Furthermore, in a sensitive and functional bioassay, a NP labelled biomolecule must retain its specific binding to analyte, while keeping the non-specific adsorption (NSA) on the substrate to minimum. Therefore the linker must facilitate this requirement by being a linker specific for a particular functional group, for example, an amino acid functional group on a biomolecule.
Clearly there is a lack of suitable biomolecular nanoparticle based label systems which are not prone to aggregation, show good non-specific binding, high selectivity and which can bind and maintain the active form of the protein.
Thus, it is desirable to provide improved bioanalytical systems, in which bioassays use superior labels comprising multivalent linkers capable of linking the biomolecular detection species to the nanoparticle label in a way that will not result in a reduction in activity, and which not compromise a dispersion of the nanoparticles. Desirably, the improved label will lead to high assay sensitivity and ultralow LODs for a desired analyte.
Summary of the Invention
According to the present invention, as set out in the appended claims, there is provided a label comprising:
a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a sta bil izing effect on the a bsolute value of the zeta potential at the nanoparticle surface.
Alternatively, the dendrimer may conjugate with one or more intermediate molecules that in turn may conjugate with the target analyte.
The sta bil izing effect is relative to the a bsol ute value of the zeta potential, as the zeta potential can be positive or negative depending on the overal l charge present on the nanoparticle. I n one em bodiment, the sta bilizing effect results from an increase in the a bsol ute value of the zeta potential at the nanoparticle su rface. I n prior art la bels, addition of an uncharged l in ker, for example, an succinimidyl ester l inker or a gl utaraldehyde linker, to the nanoparticle surface leads to a drop in the a bsolute zeta potential, as the uncharged linker becomes attached to the surface. The dendrimer functionalised nanoparticles of the invention of the invention can be directly conjugated with the target analyte without need for such neutral linking agents which undesira bly dilute the charge on the nanoparticle surface. In other words, the electrical sta bility of the nanoparticle is adversely affected desta bil ising nanoparticle colloids and increasing the tendency towa rds nanoparticle aggregation . The dendrimer linkers overcome this problem since they typical ly have ionisa ble chemistries, for example, carboxylate or amine chemistries, wh ich can be manipulated to sta bil ize the charge on the nanoparticle. H igh zeta potential wil l confer particle sta bil ity and accordingly, the colloidal dispersion of the nanoparticles wil l resist aggregation. When the zeta potential is low, attraction exceeds repulsion and the dispersion wil l break and the particles aggregate. Because a zeta potential can be positive or negative, an increase in zeta potential within the present invention is an increase in the a bsolute val ue thereof.
Dendrimers of the present invention will at least not desta bilize a zeta potential (lower the a bsol ute valve). A zeta potential having an a bsol ute val ue in mV of from a bout 20 and greater is desira ble (a value that is greater than +20 or less than -20 is desira ble). Suita bly the zeta potential has an a bsolute val ue of a bout 25 or greater such as a bout 30 or greater. It will be appreciated that the positive effect observed on the a bsolute zeta potential by conjugation of the dendrimers of the invention to a nanoparticle surface may also be considered as a non-desta bilizing effect. Desira bly when the dend rimer is linked to the nanoparticle there is an increase in the zeta potential that offsets any greater tendency of a dispersion of the dendrimer-nanoparticles to come out of their dispersed state. For example it may be desira ble that an increase of greater than a bout 30%, desira bly greater than a bout 35%, such as a bout 40%m in the zeta potential is achieved as compared to that of the non-functionalized nanoparticle.
Desira bly, the fu nctional group provided on the dendrimer may be a carboxylate or an amine functional group (the nanoparticle needs to have a corresponding group on its surface, for example, amine or carboxylate, respectively). Prefera bly, the functional group on the dend rimer is a carboxylate group (-COOH ).
The fu nctional grou ps provided on the nanoparticle may be phosphate, amine or carboxylate functional groups or com binations thereof. The nature of the groups are such that they complement each other, for example, where the dendrimer comprises an activata ble carboxylate group, the nanoparticle will be provided with a proportion of amine functional groups to allow dendrimer binding to the nanoparticle surface. On the other hand, where activata ble carboxylate functional groups are provided on the nanoparticle, the dendrimer may suita bly comprise amine groups. However, it is preferred to provide a system where the nanoparticle surface functionality is predominately negatively charged with a smal l fraction being functional ised with amine for suita ble linking to carboxylate terminated dendrimers.
The skil led person will appreciate at physiological pH, at which biological assays are typical ly carried out, carboxylate and phosphate groups are unprotonated and thus carry negative charge, while amino groups are protonated and wil l be positively charged.
In one particular aspect of the invention, activation methods for nanoparticles using dendrimers as linkers that feature very high specific binding and very low non-specific binding are provided. For example, in preferred em bod iments, the dend rimer may be terminated with carboxylate (-COOH ) chemistries which can be activated by sulfo N-hydroxysuccinimide and a dehydrating agent such as l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, for exa mple. Activation of the dendrimer -COOH groups is desira ble, since the overal l negative zeta potential of the nanoparticle is maintained, al lowing the nanoparticles to retain colloidal sta bil ity and increases the efficiency of the reaction with biomolecular target of interest. Activation of -COOH groups of the dendrimer with sulfo-N HS results in a formation of sulfo-N HS-ester that spontaneously react with amino-groups of the target antibody. Sulfo-N HS-esters advantageously longer lived than N HS-esters, possess a negative charge and so facilitate production of a more stable product of thermodynamic control. This allows the labels of the invention to bind more active proteins on the nanoparticle surface. Thus, the invention provides an improved ultra sensitive la bel having an optimised l in kage between the nanoparticle la bel and the biological ly relevant analyte molecules of interest, while retaining the negative electrostatic charge on the nanoparticle, thus preventing aggregation . The nanoparticle can then be derivatized for detection using methods known to persons skilled in the art.
Prefera bly, the nanoparticle surface fuctionalisation comprises a first ionisa ble functional group wh ich is inert to a target analyte and a second ion isa ble group functional group for immo bilising at least one dendrimer onto the nanoparticle surface, wherein the first and second ionisa ble functional groups have a different electrostatic charge and the second ionisa ble functional group is present on the nanoparticle surface in an amount sufficient to prevent nanoparticle aggregation in a col loid sol ution. Prefera bly, the dendrimer is preassem bled or is partial ly assem bled before it is immo bil ised onto the nanoparticle surface.
Thus, to ensure colloidal stability is maintained, the nanoparticle surface must be appropriately treated prior to dendrimer attachment. Desirably, the majority of the surface of the nanoparticles may be provided with, for example, negatively charged phosphate groups which provide a base of negative charge. The phosphate groups are advantageously inert towards proteins and desirably greatly reduce non-specific binding of non target molecules. This leads to labels having high selectivities and allows for very high assay precision. Preferably, the first ionisa ble function group is present on the nanoparticle surface in a range of from a bout 87% to 93% of the total nanoparticle su rface area. Suitably, from about 3% to about 20% of the nanoparticle surface may be provided with reactive amino (-N H2) groups which facilitate attachment of carboxylate (-COOH) terminated dendrimer. More preferably still, the nanoparticle surface may be provided with from about 5% to about 15% amino (N H2) groups for dendrimer attachment. Suitably, the nanoparticle surface is provided with from about 7% to about 13% of amino (N H2) groups onto which the -COOH terminated dendrimer could be attached. Most preferably, the surface is provided with from about 9% to about 11% of amino (-N H2) groups for dendrimer attachment. The amount of surface coverage may be determined by methods known to the skilled person, for example, ninhydrin assay, wherein the nanoparticles are reacted with a known concentration of ninhydrin which binds to the -N H2 groups on the nanoparticles and causes a quantifiable colour change which can be detected by UV/fluorescent methods and measured against a calibration curve,
It is important for colloid stability and maintenance of high absolute zeta potential that the nanoparticle surface is functionalised with predominantly a functional group of one charge wherein this group is inert to the target analyte and that a minimum portion of the nanoparticle surface is functionalised with a functional group of an opposite charge, wherein this latter functional group present in minimal quantity is used to tether or immobilize dendrimer to the nanoparticle. For example, ideally a nanoparticle where a negative functional group which is inert to the target predominates on the nanoparticle surface, with a minimum but sufficient amount of a positively charged group is present to bind dendrimer through carboxylate groups on the dendrimers. The advantage of such a system is that the presence of immobilized -COOH terminated dendrimer on the negative nanoshell enhances significantly the zeta potential by increasing and stabilizing the negative charge at the nanoparticle surface. The inert functional groups do not react with the target analyte and so nonspecific binding is very low. Critically, the inventors have found that increasing the percentage of the amino group on the surface of the nanoparticle to over about 20% surface coverage results in immediate aggregation of the nanoparticles. Accordingly, the combination of negatively charged surface phosphate groups with a relatively low % of surface coverage with amino groups and less than about 20% amino coverage, provide negatively charged nanoparticles which can react with colloid stabilizing negatively charged carboxylate terminated dendrimers to provide labels that allow for very high target specific binding with low non-specific binding. The negatively charged dendrimer enhances and increases the zeta potential of the nanoparticle, increasing the resistance to agglomeration in a colloid solution.
Accordingly, in one aspect there is provided a label comprising:
a nanoparticle wherein the nanoparticle surface is functionalised with
(i) a first ionisable functional group which is inert to a target analyte;
(ii) a second ionisable functional group for immobilising at least one dendrimer onto the nanoparticle surface;
said dendrimer having at least one activatable functional group for conjugating the target analyte to the functionalised nanoparticle, the dendrimer has a stabilizing effect on the zeta potential at the nanoparticle surface, and
wherein the first and second ionisable functional groups have a different electrostatic charge and the second ionisable functional group is present on the nanoparticle surface in a amount sufficient to prevent nanoparticle aggregation in a colloidal solution.
In other words, there is provided a label comprising
a nanoparticle functionalised with a proportion of charged functional groups and a proportion of charged functional groups of opposite charge,
one of said charged groups being predominant to prevent agglomeration of the nanoparticle while being inert to a target and the other being sufficient to capture the target but not destabilize to cause agglomeration.
The first ionisable functional group which is inert to a target analyte predominates on the nanoparticle surface and set a basic charge. Preferably, the basic charge is negative. Preferably, the first ionisable functional group is -COOH. Preferably, the second ionisable functional group is - NH2.
Thus, the labels of the invention advantageously allows for assays with improved label stability, target binding, assay sensitivity, and LOD of the assay compared to the results achieved using alternative linkers such as neutral linkers. The nanoparticle label can easily be prepared while maintaining the integrity of a colloid dispersion of the nanoparticles.
The conjugation method of the present invention utilises dendrimers as multivalent linkers and result in a surprising improvement in attachment of biomolecules onto the surface of NPs. The conjugation method disclosed herein advantageously provides stable colloids, as well as increasing the active surface area of the particle. Furthermore, the special dendrimer -COOH activation method using EDC/NHS or sulfoNHS activation allows the label to bind to and retain target protein activity. This means when the label is used in sandwich assays with an antigen for example, the antigen retains its activity towards antibody despite the label binding reaction. This is advantageous since it allows for high sensitivity and low LOD/LOQ in the assay. Use of neutral linker can deactivate protein recognition ability.
As explained above, conjugation of the analyte to the nanoparticle by a dendrimer linker of the invention has the desirable effect of increasing the zeta potential at the nanoparticle surface. This may be the absolute zeta potential at the nanoparticle surface. These may be groups that are binding groups for binding other moieties to the nanoparticle. For example, these may be non-active groups that have been treated to make them active for bonding. For example, the dendrimer may already have, or may already be, functionalised with groups suitable to bind the dendrimer to a nanoparticle and/or target analyte. Groups that allow such binding will be referred to herein as an "activated" or "activatable" group. Typically, such activated or activatable groups include carboxylate or amine groups, which may be activated by methods known in the art. The activation generally renders the groups more reactive towards other corresponding groups, for example, amine or carboxylate groups on other moieties. The dendrimer may need to be activated in order to conjugate it to the nanoparticle surface and/or to conjugate the dendrimer to an amino acid on a protein, a cell, antibody, antigen or other suitable analyte. Desirably, the activated groups on the dendrimer are susceptible to hydrolysis. Hydrolysis can be employed to form charged groups at physiological pH. Therefore, the electrostatic charge on the nanoparticle is increased and colloidal stability is maintained, despite the modification of the surface. The invention is an improvement over prior art nanoparticle labels which require use of linkers, which when bound to the nanoparticle surface, can detrimentally affect the zeta potential of the nanoparticle. For this reason, prior art nanoparticle labels suffer from problems such as nanoparticle aggregation.
Accordingly, the invention provides a label comprising a nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a target analyte to the nanoparticle. A dispersion of dendrimer functionalised nanoparticles according to the invention will desirably have a zeta potential which is increased as a result of the conjugation of dendrimer to the nanoparticle surface. An increased zeta potential can offset a greater tendency of nanoparticles that are larger (by virtue of surface modification) to agglomerate. Such a stabilizing effect on the colloidal dispersion of the nanoparticles is desirable since undesirable aggregation or flocculation of the nanoparticles is hindered. This avoids problems that may occur during an assay, for example, particles "dropping out" of solution and too slow a flow rate where lateral flow microfluidic devices are used for analysis.
Furthermore, use of dendrimer linkers, having a plurality of reactive groups, improves the probability of binding of the analyte of interest to the nanoparticle. This increases the reaction yield and surface coverage to provide the basis for a surprisingly sensitive analytic technique which can be used to detect unexpectedly low levels of analyte.
In a related embodiment, there is provided a method for preparing a label comprising the steps of:
(i) functionalising a nanoparticle surface with a first ionisable functional group which is inert to a target analyte and with a second ionisable functional group for immobilising at least one dendrimer onto the nanoparticle surface, wherein the first and second ionisable functional groups have a different electrostatic charge and the second ionisable functional group is present on the nanoparticle surface in a amount sufficient to prevent nanoparticle aggregation in a colloidal solution ;
(ii) immobilising a dendrimer onto the nanoparticle surface by reacting the second ionisable functional group with a suitable functional group on the dendrimer, the dendrimer having at least one activatable functional group for conjugating a target analyte and wherein the dendrimer is terminated with a third functional group of the same charge as a first ionisable functional group on the nanoparticle and wherein the third functional group is reactive towards the target analyte.
In a preferred embodiment, the activatable functional group of the dendrimer is a carboxylate.
In a preferred embodiment the first ionisable functional group is phosphate.
In a preferred embodiment the second ionisable functional group is -N H2.
In a preferred embodiment the third functional group is -COOH.
In one embodiment, the dendrimer functionalised nanoparticle may be conjugated to a detection molecule, which in turn detects a target analyte. The detection molecule may be highly selective for a corresponding target analyte. By highly selective it is meant that the selectivity for a moiety approaches near specific.
Accordingly, in one aspect of the invention, the label of the invention comprises a functionalised nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a detection molecule which is highly selective for a further target analyte thereto, wherein the dendrimer is selected to increase the zeta potential of the nanoparticle. The label comprising the detection molecule can then be used to bind a further analyte of interest in a highly selective manner. The detection molecule in this regard may be highly selective for the further analyte of interest. An example would be use of an antibody as a detection molecule (detection antibody) which is (highly) selective for an antigen (capture molecule) which is the target analyte of interest. It will be appreciated that the detection molecule may be an antigen, for which a particular anti body (capture molecule) is the target analyte. Similarly, a particular antibody used as a detection molecule, may be used to detect a particular cel l expressing a particular antigenic part of a protein.
Accordingly, the la bels of the invention are useful tools for biological discovery and biomed ical detection, medical imaging and therapeutic appl ications such as cel l la bel l ing, targeted drug del ivery, targeted gene del ivery, biosensing, cel l separation, cell purification a nd imaging.
Dendrimers are monodispersed compounds with a well -defined structure, in which the num ber and functional ity of the dendrimer functional groups can be tailored to the h ighly selective bioconjugation required. Use of multivalent or multifunctional dendrimers as linkers in bioconjugated nanoparticle la bels leads to a higher efficiency in bioconjugation of, for example, ta rget analytes (e.g., antibod ies or antigens), and overall facil itate an improved assay performance with ultra low LO D capa bility and in a highly sensitive and precise manner. Binding occurs more rapidly and so assays are completed in less time. These are attractive advantages, since such improvements are necessary for reducing costs in medical d iagnostics, particularly where large num bers of samples are tested. The resu lts obtained using the la bels of the invention are also consistent. They do not vary depending on the batch of la bels used.
Greater surface coverage and n umber of availa ble binding sites for a target analyte leads to more efficient bioconjugation. Generally, the rate of the bioconjugation reaction will be proportional to the concentration of the reactants. I n the case of the present invention, dendrimers provide more reactive groups and therefore a higher surface concentration, than would likely be achieva ble with other monovalent linkers that don't have multipl icity. Rates of bioconjugation of the target analyte to the nanoparticle la bel are improved. Furthermore, the multivalency of the dendrimer increases the probability that analyte will bind to the nanoparticle surface. These factors lead to superior nanoparticle label performance in assays.
The invention also relates to a dispersion of labels of the present invention in a suitable medium, desirably an aqueous medium.
I n a preferred em bod iment, the dendrimer is a PAMAM dendrimer or a Newkome dend rimer. Suitably, the hydrophilic dendrimer is a PAMAM dendrimer. PAMAM dendrimers are quite water-soluble. Newkome dendrimers may require sonication to assist in dissolving them in water. In an embodiment where Newkome type dendrimers are used, generation 1 dendrimer are preferred as the full generation Newkome dendrimer presents carboxylate (-COOH) terminal functional groups. In an embodiment where PAMAM dendrimers are used, half-generation dendrimers are preferred as the half generation PAMAM dendrimers presents carboxylate (-COOH) terminal functional groups which are the most preferred dendrimer terminating groups of the invention. I n a preferred em bodiment, the dendrimer is at least one dendrimer selected from the group consisting of generation 0 to generation 10 dendrimers, incl uding half generations of dendrimers. Most preferred are generations that provide carboxylate (-COO H) terminal chemistries. Desira bly, the dendrimer is a generation 0 dendrimer, a generation 0.5 dendrimer, a generation 1 dendrimer, a generation 1.5 dendrimer, a generation 2 dendrimer, a generation 2.5 dendrimer, a generation 3 dendrimer, a generation 3.5 dendrimer, a generation 4 dendrimer, a generation 4.5 dendrimer, a generation 5 dendrimer, a generation 5.5 dend rimer, a generation 6 dendrimer or a generation 6.5 dendrimer. More prefera bly, the generation size of the dendrimer may be from generation 0 to generation 4.5. The most preferred size is generation 4.5. Dendrimers of generation 4.5 or smal ler are more desira ble since, the cost of the higher generations is significant and accordingly they are not ideally suited for use in an inexpensive disposa ble diagnostic device. Generation G4.5 dendrimers typically provide an optimum balance between cost and performance.
Prefera bly, the dendrimer is of a generation or half generation which is carboxylate (-COOH ) terminated.
I n a particula rly preferred em bodiment, the dendrimer is a generation 4 dendrimer. In another preferred em bodiment, the dendrimer is a generation 0 dend rimer. In yet another preferred em bodiment, the dendrimer is a generation 2 dendrimer. In another preferred embodiment still, the dendrimer is a generation 3 dendrimer.
I n one em bod iment, the dendrimer is a hydrophil ic or a hydrophobic dendrimer. Desira bly, the dend rimer is a hydrophilic dendrimer, since a num ber of hydrophobic dend rimers (incl uding some starburst dendrimers) are comprised of conjugated aromatic rings. A num ber of these dendrimers may present water sol u bility issues.
It wil l be appreciated that depending on the surface fuctionalisation of the nanoparticle, the dendrimer and the nanoparticle must have compati ble surface chemistries. For example, preferred nanoparticles may comprise non-functional ised surfaces such as silica surfaces. However, the silica surfaces of the nanoparticles of the invention are typical ly functional ised with groups necessary to facilitate conjugation to the dendrimer. For example, the nanoparticles may be functionalised with amine groups. Accordingly, the dendrimer l in ker should comprise linking functionalities, for example, carboxylate surface chemistries (or other functional ities capa ble of l in king to amine) so that the groups may react together to l ink the dendrimer to the nanoparticle surface. Examples of dendrimers having carboxylate surfaces include whole generation, such as generation 0, generation 1, generation 2, etc., or PAMAM type dendrimers of half generation, e.g., generation 0.5, generation 1.5, generation 2.5 etc. It wil l also be appreciated that if the nanoparticle surfaces comprise surface carboxylate grou ps, then dendrimers using amine surface chemistries may be used. Prefera bly, the la bel of the invention comprises a non-magnetic, non-metal nanoparticle, such as a sil ica nanoparticle. Thus, in a preferred em bod iment, the nanoparticle may be a silica nanoparticle or a metal oxide nanoparticle. Suita bly, the nanoparticle may be a sil ica particle. Silica is preferred in many appl ications since it is derivatisa ble with bioreactive functional groups, is biocompatible, wh ile being a relatively inert su bsta nce that prevents agglomeration with other nanoparticles in a dispersion .
Preferably, the label of the invention comprises a silica nanoparticle functionalized with at least one dendrimer for conjugating a target analyte to the nanoparticle, so as to allow labelling of the analyte for detection thereof, wherein the dendrimer linker is selected so as to increase the zeta potential of the nanoparticles in a dispersion of the nanoparticles. I n a preferred embodiment, nanoparticle may be at least one of dye-doped, magnetic, sil ica or metal doped. I n a preferred em bodiment, the nanoparticle is may be a hybrid nanoparticle comprised of a hybrid material and include, noble metal, q uantum dot or magnetite doped silica nanoparticles. Suita bly, the nanoparticle may be a dye-doped nanoparticle which advantageously provides for fluorescence detection. Desira bly, where the nanoparticle is a dye-doped nanoparticle, the dye-doped nanoparticle may be fluorescence optimized, for example, for near infrared ( N I R) electromagnetic radiation. This makes the la bel detecta ble by spectrophotometric techniques.
I n a particularly preferred em bodiment, the nanoparticle is a dye-doped sil ica nanoparticle. Near-infrared dye doped sil ica N Ps, in which the fl uorescence has been optimized are preferred since at near infrared wavelengths there is low background interference from the fluorescence of biological molecules, solvent, and su bstrates. Furthermore, whole blood has a weak a bsorption in the N I R region, thus reducing the need for whole blood filtering for assays using whole blood . N I R l ight can a lso penetrate skin and tissue to several mill imetres ena bl ing fluorescence detection in vivo, for example, in dermatological or in-vivo diagnostic devices.
I n a preferred em bodiment, the nanoparticle may have a diameter in the range of a bout 10 nm to a bout 1000 nanometres. The size of the nanoparticle used will depend on the intended appl ication and the size of the target analyte in question. Smal ler nanoparticles may used for assay involving smal l proteins or nucleic acids, whereas larger nanoparticle la bels may be used for appl ication involving cel ls, for example.
The silica surface of the nanoparticle may be functionalised with molecules comprising reactive groups for example at least one one of silane, amines, thiols, phosphates, epoxy, carboxyl, cyano, isothiolcyanate, iodoacetamide, azido, NHS-ester groups. Suitably the molecule comprises at least one organosilane and desira bly comprises an organo group comprising at least one of thiol, amine or carboxylate, phosphate, epoxide or isothiocynate functional groups. Desirably, the surface layer further comprises at least one of a generation 0 to a generation 10 dendrimer and half generations thereof and which may be carboxylate or amine terminated depending on the appl ication. Carboxylate dendrimer chemistries are preferred since they are activatable towards biotargets without the need for use of neutral linkers.
It will be appreciated that the nanoparticle surface is modified to comprise desired functional groups. For example, a suitable organosilane may provide reactive groups, for example, amine functionalities on the surface of the nanoparticle which are suitable for binding to dendrimer having activated (or activatable) carboxylate chemistries. In addition to such silanes, it is desirable to provide phosphonate groups having negative charges on the nanoparticle surface; such negatively charged phosphonate groups are necessary to stabilize the nanoparticle. An example of a phosphonate providing organosilane is trihydroxypropylsilanemethylphosphonate. The labels of the invention may comprise nanoparticles having such functionalised silica surfaces, wherein the reactive groups may be selected from the group of activatable biochemical groups consisting of: thiol, amine or carboxylate, phosphate, epoxide or isothiocynate. Preferably, a mixture of phosphate and amine groups are provided on the nanoparticle surface in the ranges discussed previously above. The functionalizing group or groups will depend on the dendrimer linker required and/or other molecules of interest that can be added to the nanoparticles for various reasons. Suitably, the nanoparticle of the invention may be further functionalised with at least one of a protein, a nucleic acid, or a molecular label, a fluorophore, a nanoparticle or other moiety having a suitable group for binding to a functional group on a surface modified nanoparticle, for example, amine or carboxylic acid. In one embodiment, the label of the invention may comprise a nanoparticle having a modified silica surface comprising phosphate, amine or carboxylic groups and combinations thereof, and a plurality of dendrimers. Such dendrimers may be all of the same generation or half generation, or may be of different generations, depending on the application. The surface layer may be further modified to comprise at least one of a protein, a nucleic acid, a molecular label, a molecular tag, a fluorophore or a nanoparticle. Suitably, the modified silica surface layer may have a thickness in the range of about 1 nm to about 100 nanometres.
In a preferred embodiment, the target analyte may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte highly selective for a further analyte species.
As mentioned above, a detection molecule highly selective for target analytes can be employed. An example of a detection molecule is a protein, such as an antibody, an antigen, or other moiety having a group suitable for attachment to a dendrimer of the invention, which may be used to selectively bind to a target analyte (for example, antibody, antigen) in an immunoassay. Desirably, the detection molecule is highly selective for a target analyte selected from the group consisting of: a cell, a pathogen, a protein, a secondary analyte, a molecular label, a molecular tag, and a nucleic acid.
By secondary analyte it is meant a moiety that may selectively bind to the target analyte. The secondary analyte may then be probed for, or detected by a detection molecule that is highly selective for the secondary analyte. This is useful where a detection molecule highly selective for the target analyte is not available.
Suitably, the target analyte is a protein, for example, green fluorescent protein. In a particularly preferred embodiment, the protein may be an antibody, such as a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen. Desirably, the detection molecule may be a monoclonal antibody, a polyclonal antibody or an antigen. Suitably, the antibody is a detection antibody which is highly selective for a target analyte.
By molecular tag is it meant a peptide sequence which is removeably grafted onto a recombinant protein for reasons including facilitating protein detection in assays, protein purification, etc. By molecular label it is meant a chemical moiety with a detectable property, for example, a fluorescent or radioactive molecules which can be attached to a nucleic acid as a detectable group.
In a preferred embodiment, the molecular tag may be biton or streptavdin.
In a preferred embodiment, the molecular label may be a chemosensor, a fluorescent or a radioactive probe. Suitably the fluorescent probe is green fluorescent protein.
In a particularly preferred embodiment, the nucleic acid is a DNA or an NA.
In a preferred embodiment, the target analyte, the detection molecule or the secondary analyte may be a protein, including a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen.
Accordingly, in a related aspect the label of the invention comprises a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a detection molecule which is highly selective for a corresponding target analyte, wherein the dendrimer increases the zeta potential of the nanoparticle.
In a preferred embodiment the detection molecule is a monoclonal antibody, a polyclonal antibody or an antigen.
Desirably, the antibody may be goat antihuman IgG. In a preferred embodiment, the antigen may be polyclonal human IgG.ln another aspect of the invention, there is provided a method of preparing a nanoparticle la bel for detecting a target analyte comprising the steps of: (i) activating at least one functional group on a provided nanoparticle to provide at least one activated functional group having enhanced reactivity; and
(i) condensing the at least one activated functional group with a functional group of a provided dendrimer to link the dendrimer to the nanoparticle; and optionally, (ii) quenching the at least one activated function group.
Suitably, the label may be reactivated before use. Desirably, the method may further comprise the step of attaching at least one detection molecule to the activated label to form a label. Preferably, the detection molecule is highly selective for a target analyte. Desirably, the nanoparticle may be a dye-doped nanoparticle or a magnetic doped nanoparticle. It is preferred that dye-doped nanoparticles are used.
The nanoparticle label formed will comprise an activated (or activatable, if quenched after initial preparation) dendrimer linked to a nanoparticle. In other words, an activated label is formed. The thus formed activated label may be used immediately in an assay. Alternatively, the activated label may be quenched and stored until required for use. The functional groups on the dendrimer may be activated at a later time, when required for use in an assay or other application.
In one aspect of the invention the label of the invention may be used in an immunoassay. Preferably, the immunoassay is an immunosorbent assay, such as an ELISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.
Desirably, the label of the invention may be used as a tool for biological discovery and/or biomedical detection, in medical imaging and/or therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.
Suitably, the label of the invention may be used in a dermatological or in vivo diagnostic device.
In one embodiment, there is provided a method of detecting a target analyte in a test substance using the label of the invention comprising the steps of:
(i) activating at least one of the activatable functional groups on the dendrimer to form an activated label; and
(ii) exposing the activated label to the test substance comprising the target analyte; and
(iii) analysing the test substance for bound label. Preferably, the test substance is exposed to the activated label under conditions that allow the activated functional group to bind to the target analyte.
In a preferred embodiment, the label of the invention may be used in an immunoassay. By immunoassay, it is meant an assay which measures the concentration of a biochemical substance in a biological liquid, such as serum or urine. Such assays are generally based on the reaction of an antibody to an antigen.
Accordingly, in a related aspect of the invention there is provided a method of directly detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of:
(i) immobilising a target analyte onto a support;
(ii) washing the support to remove unbound target analyte;
(iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
(iv) washing the support to remove unbound activated label; and
(v) analyzing for bound target analyte.
Suitably, the target analyte may be may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte which is highly selective for a further analyte species.
Suitably, the immunoassay may be an immunosorbent assay, an ELISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.
It is preferably that the immunosorbent assay is a plate-based assays for detecting and quantifying biochemical substances, for example, peptides, proteins, antibodies and hormones, etc.
Suitably, the target analyte may then be directly detected by exposing it to a suitable label of the invention, for example a nanoparticle label having dendrimer linked to a primary antibody, which is highly selective for the target analyte. Alternatively, the target analyte may be indirectly detected, by exposing the target analyte to a secondary analyte molecule (for example, a primary antibody which is highly selective for a target antigen) before using a label of the invention comprising a primary antibody as a detection molecule that is highly selective to the secondary analyte molecule (nanoparticle label having dendrimer linked to a detection molecule (for example, primary antibody) which is highly selective for the secondary analyte molecule. Immunosorbent assays do not usually use direct detection methods. Desirably, instead, a detection molecule (for example, a secondary antibody) that has been linked to a nanoparticle label is used to probe for a secondary analyte molecule (for example, a primary antibody which is highly selective for the target analyte), which is bound to the target analyte. Thus, the target analyte is detected indirectly through by probing with the detection molecule for a secondary analyte.
Alternatively, another form of indirect detection may suitably be used involves using a primary or secondary antibody that is labelled with an affinity tag such as biotin. Then a secondary (or tertiary) probe, such as streptavidin that is labelled with the nanoparticle label of the invention, can be used to probe for the biotin tag to yield a detectable signal.
Accordingly, in a related embodiment, there is provided a method of detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of:
(i) immobilising a target analyte onto a support;
(ii) washing the support to remove unbound target analyte;
(iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
(iv) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule;
(vi) exposing the activated label to the support so that the detection molecule binds to the secondary analyte molecule;
(vii) washing the support to remove unbound activated label; and
(viii) analyzing the support for bound label.
Suitably, in the immunosorbent assay, the target analyte (for example, an antigen) may be immobilized onto a support. Desirably, the support is a microplate or a plurality of polystyrene beads or a gel film or the like. Suitably, the immunosorbent assay may be performed in 96-well (or 384-well) polystyrene plates, which immobilize antibodies and proteins, including antigen. Using a plate assists in the separation of the bound material from the unbound material during the assay. Advantageously, unbound material may be washed away to facilitate the assay.
Suitably, the target analyte may be a protein, a detection molecule or a secondary analyte which is selective for a further analyte species. Preferably, the detection molecule and/or the secondary analyte is an antibody. It will be appreciated that an antibody that recognizes the target analyte is called the "primary antibody". It will be further appreciated that if the primary antibody is linked to a nanoparticle label of the invention, then direct detection of the analyte is possible. This technique is useful for immunohistochemical staining of tissues and cells. Advantageously, the target analyte may then be complexed with the detection molecule (for example, an antibody), which then forms part of the label of the invention.
Suitably, the immunosorbent assay is a sandwich assay. This is desirable since it is one of the most powerful immunosorbent assay formats. This is a type of capture assay which is called a "sandwich" assay because the target analyte to be measured is bound between two primary antibodies, i.e., the capture antibody which is used to immobilize the target analyte onto a support and the label of the invention which comprises a detection antibody which is selective for the target analyte. The sandwich format is preferred since it is a sensitive and robust technique.
In a related embodiment, there is provided a method of detecting a target analyte in a sandwich immunosorbent assay using the label of the invention comprising the steps of:
(i) immobilising a target analyte onto a support;
(ii) washing the support to remove unbound target analyte;
(iii) exposing the support to a secondary analyte molecule which binds to the target analyte;
(iv) washing the support to remove any unbound secondary analyte molecule
(iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
(v) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule;
(vi) washing the support to remove unbound activated label;
(vii) analyzing for bound label.
This type of method involves an indirect immunosorbent assay procedure in which the analyte is detected, by actual detection of a secondary analyte, which is probed for, by the detection molecule of the label of the invention.
The key step of the assay is the immobilization of the target analyte to the support phase (for example, an antigen). Desirably, immobilization of the target analyte can be accomplished by direct adsorption to the assay plate. Alternatively, the target analyte may be immobilized onto the support phase indirectly through use of a capture molecule (for example, an antibody) that has been attached to the plate before it is exposed to the target analyte.
Accordingly, if desired, in the methods of the invention described herein, the first step of immobilising the target analyte onto the support may comprise:
(i) immobilising a capture molecule selective for the target analyte onto the support; (ii) washing the support to remove unbound capture molecule; and
(iii) exposing the support to the target analyte to immobilise the target analyte onto the immobilised capture molecule.
The method involves directly detects the target analyte through an immunosorbent assay, where the target analyte is bound to a capture molecule which is itself immobilised on the support. Suitably, the capture molecule may be an antibody or an antigen.
In all of the assay methods described herein involve a step of providing a nanoparticle label comprising at least one activatable dendrimer for linking the label to a target analyte, the dendrimer comprising at least one activatable functional group; and activating at least one of the activatable functional groups on the dendrimer to form an activated label. The activated label may then be immediately used in an assay.
Desirably, the methods may be carried out under conditions that allow the activated functional group of the dendrimer on the activated label to bind to the target analyte or to the secondary detection molecule. The target analyte therefore links to the label through the detection molecule or through the secondary analyte as the case may be. The test substance may then be analysed for target analyte bound nanoparticle.
In a particularly preferred embodiment, the nanoparticle label, such as a dye-doped nanoparticle. In a preferred embodiment, the target analyte may be protein, a nucleic acid, an antibody or an antigen. Suitably the target analyte is a protein, such as GFP. Preferably, the target analyte is an antigen, such as human IgG. Desirably, the capture molecule is an antibody, such as goat antihuman IgG. It is preferred that the detection molecule is an antibody, such as goat antihuman IgG. Suitably, the test substance may be tissue, blood or a tissue or blood derivate. Preferable the support is a micro plate or a plurality of polystyrene beads. Desirably, the support is washed with a buffer, such as N-morpholinoethylsulfonic acid (MES) or Phosphate buffer saline (PBS), polysorbate materials (including polysorbate 20 and 80 such as those sold under the brand name Tween®), sodium dodecyl sulphate. Advantageously, the test substance may be tissue, blood, for example, whole blood or a blood or tissue derivate. Suitably, the test su bstance sample may be worked up (cleaned up) before assay.
In a particularly preferred embodiment, the activating step necessary to form an activated label involves treating the nanoparticle label (comprising activata ble dendrimer, or activatable carboxylate groups on the nanoparticle, if provide in this order) with N-hydroxysuccinimide (N HS) or sulfo N-hydroxysuccinimide, and a dehydrating agent. Sulfo N-hydroxysuccinimide and a dehydrating agent are the preferred activating reagents, since use of sulfo N-hydroxysuccinimide provides a more stable solution of the nanoparticle conjugates. Suitably, the dehydrating agent may be l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC). This activating system is capable of activating carboxylate groups on the dendrimer to enhance their reactivity towards amine groups on the nanoparticle surface, (or vice versa, where the nanoparticle comprises activatable carboxylic acid groups and the dendrimer comprises amine surface functionalities). It will be appreciated that other activating system may be suitably used to activate other types of functional groups.
Desirably, the nanoparticle label may be activated by treating the nanoparticle label with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC). In this embodiment, the dendrimer carboxylate functional groups are made more reactive towards amines groups. Amine groups can be found on the nanoparticle surface. Amine groups can also be found on suitable target analyte or detection molecules. The reaction may take place in a MES buffer. Desirably, the buffer may have a concentration in the range about ImM to about 1M. Suitably, the pH of the buffer may be in the range of about 3 to about 9. Desirably, the reaction may proceed for an activation period in the range about 10 seconds to about 30 minutes.
In embodiments where the dendrimer has a carboxylate surface chemistry, particular advantages arises from activating the carboxylate groups with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) in 0.1 M MES buffer, at pH = 6.0, for 30 minutes. Desirably, the ratio of EDC:NHS may be 4:1. Preferably, the N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide may be present in a two fold molar excess of NHS reagent per carboxylate group (2:1). These conditions result in conversion of dendrimer carboxylate groups to semi-stable reactive NHS- esters. Typically those reactive NHS-esters have a maximum lifetime of about 60 minutes, an often of about 30 minutes. The availability and the lifetime of the NHS esters on the dendritic linker will determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labelled. In a preferred embodiment of the invention, the label comprises activated dendrimer surface modified nanoparticle comprises semi-stable amine reactive NHS-esters.
It is noteworthy to mention that at these conditions, the optimum balance between the rate of aminolysis and the hydrolysis of the dendritic N-succinimidyl esters was observed. This is critical for maintaining high activity and specificity of a conjugated target capture antibody towards the antigen in an immunoassay, for example.
The dendrimers of the nanoparticle label of the invention may be activated more than one. This is desirable, since the dendrimer label may be pre-prepared and sold in preformed form according to application. When required for use, the dendrimer can be activated in the laboratory. There is also provided a kit for testing for target analyte, the kit comprising:
(i) functionalised nanoparticles;
(ii) activata ble dendrimer of at least one predetermined generation;
(iii) optional ly, capture molecule;
(iv) optional ly, detection molecule;
(v) optionally, support;
(vi) instructions for use.
The kit of the invention may further comprise activating reagents. Desira bly, the kit may further comprise buffer and/or wash solutions.
Brief Description of the Drawings
The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:-
Figure 1 shows examples of the monovalent and multivalent cross-linkers used in this work; Figure 2 shows a micrograph of dye-doped silica nanoparticles;
Figure 3 shows dynamic l ight scattering ( DLS) data showing the amount of nanoparticle aggregation (A) as effect of the corresponding l in ker attached to its surface and ( B) change in zeta potential as a function of the change on the su rface of the N I N P;
Figure 4 shows normalized fluorescence intensity measured after immobil ization of target analyte Green Fluorescent Protein (G FP) onto the G2 modified N P su rface under various cond itions;
Figure 5 shows normalized fluorescence intensity measured after immobil ization of target Green Fluorescent Protein on the N P su rface modified with sulfo-SMCC or the G2 dend rimer;
Figure 6 shows fluorescence l inked immunosorbent assays for the detection of h lgG using monovalent and bi-l inkers a nd dend rimer multivalent l inkers;
Figure 7 shows the total fluorescence signal over background fl uorescence signal, F/Fo combined with coefficient of variance, and LO D results for each l inker in the fluorescence l inked immunosorbent assay for detection of human IgG;
Figure 8 shows the rate of change in signal as target concentration changes;
Figure 9 shows comparison in the surface coverage of N I R-N Ps when using two different types of dendrimers;
Figure 10 shows binding rate of N I R-N P conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM- N P conjugates in a direct binding assay (right); Figure 11 shows the reaction parameters for the dendrimer-activated nanoparticles relative to those observed for an assay performed using antibody coupled directly to the fluorescent dye;
Figure 12 shows a schematic of the surface fuctionalisation of a preferred nanoparticle of the invention, binding to dendrimer multivalent linker and binding to target antibody.
Detailed Description of the Invention
Experimental Results
Referring now to the drawings and specifically Figures 1 to 12 inclusive and initially Figure 1. Figure 1 schematically illustrates a number of the monovalent and multivalent cross-linkers used in this work. There is provided a view of activated dendrimer depicting the activation of the carboxylic acids and the two competing reactions occurring on the activated carbonyl group. The generation 0 (GO) dendrimer is a tailor made Newkome type dendrimer having seven surface available carboxylate groups. Generation 1 and generation 2 dendrimer are commercially available Newkome type dendrimers obtained from Frontier Scientific.
Referring now to Figure 2, there is presented a micrograph of dye-doped silica nanoparticles which are approximately 71.16 +/- 3.83 nm in total diameter. The shell thickness is approximately 9.00 +/- 1-80 nm. This layer comprises an area of silica on the surface of the nanoparticle comprising trihydroxypropylsilanemethylphosphonate (for stabilising the nanoparticle) and amino groups for attachment of the dendrimer linker to nanoparticle surface.
Referring now to Figure 3A and B, and initially Figure A which shows dynamic Light
Scattering data showing the amount of nanoparticle aggregation as effect of the corresponding linker attached to its surface. It can be seen that bivalent linkers result in increased particle aggregation in comparison to dendrimer modified nanoparticles. Figure 3B shows the change in zeta potential as a function of the change on the surface of the NI RN P. The addition of the bilinker glutaraldehyde results in lowering of zeta potential relative to a nanoparticle without surface modification, and a modest increase in zeta potential where surface is modified with s-SMCC, whereas dendrimer surface modified nanoparticles show more pronounced increases in zeta potential.
Referring now to Figures 4A and 4B and initially Figure 4A in which there is presented normalized fluorescence intensity measurements taken after immobilization of target analyte Green Fluorescent Protein (GFP) onto the G2 modified N P surface under various conditions. In Figure 4A, it can be seen that by varying the stoichiometric ratio between EDC/N HS for the activation of G2 dendrimer carboxylate groups, the most target protein is bound when the ratio of EDC/N HS is 4:1. Figure 4B shows the effect of varying the activation time of the G2 dendrimer carboxylate groups before reacting with the amino fu nctional ized surface of the N Ps in the first conjugation step. It can be seen that most target protein is bound when the -COOH groups on dendrimer modified nanoparticle are allowed to activate for 30 min utes before conjugation to the nanoparticle surface takes place. Referring now to Figure 5, which shows normal ized fluorescence intensity measu red after immobilization of target Green Fl uorescent Protein on the N P surface modified with sulfo-SMCC or the G2 dendrimer. I n these reactions, both the amount of the N I R-N P and the linker (sulfo-SMCC or the G2 dendrimer) were kept constant, and the concentration of the G FP was 1.5 or 3.0 equivalents respectively ( 1 equ ivalent represents an amount of protein (in μg) that is necessary for a surface saturation of lmg of the nanoparticles). This val ue was calculated according to the formula in Reference (36). This means at the same concentration of target protein, the intensity of the fluorescent signal from the linker modified dyedoped nanoparticle is significantly higher when dendrimer is used as the l inker. This results from the fact that more of the target is a ble to bind using the dendrimer l inker that the sulfo-SMCC bil in ker and since the target is G F P, more fl uorescence is observed. This indicates that the G2 Newkome dendrimer binds more G FP per nanoparticle. Test results for G l, G2 Newkome and G 1.5 to G4.5 of PAMAM test result are presented in Figu re 9.
Referring now to Figure 6, which shows results of fl uorescence l in ked immunosorbent assays for the detection of h lgG using monovalent and bi-l inkers and dendrimer multivalent l inkers. H igher fl uorescent signal are observed when la bel comprising a dend rimer surface modified dye-doped N P is used at the same concentration of target analyte, even at lower concentrations of target human IgG. This clearly indicates that dendrimer l inkers al low a more sensitive assay.
Referring now to Figure 7, which shows the total fluorescence signal over background fl uorescence signal, F/Fo combined with coefficient of variance, and LOD results for each linker in the fl uorescence linked immunosorbent assay for detection of human IgG. It can be seen that the l imit of detection of human IgG when la bel comprising a dendrimer surface modified dyedoped N P is sign ificantly lower than the case when la bel comprising a monovalent su rface modified dyedoped N P is used, or in the case where fluorescent dye directly attaches to the antibody.
Referring now to Figure 8, which shows that rate of change in signal as target concentration changes. The drawing shows a change in the sensitivity of fluorescence for immunosorbent assay with change in concentration of hu man IgG at low concentration . No sensitivity was observed for monovalent l inkers and the organic fluorophore la bel, Cy5 at low concentrations; Signal was detected at low concentration of target when dendrimer surface modified dyedoped N P la bels were used. Referring now to Figure 9, which shows a comparison in the surface coverage of N I R-N Ps when using two different types of dendrimers; For PAMAM dendrimer coverage from 2.5% of nanoparticle surface for GO to 25% coverage for G4 dendrimer, indicating that coverage is better for h igher generations.
Referring now to Figure 10, which shows the binding rate of N I R-N P conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM-N P conjugates in a direct binding assay (right). The Figu re shows the reaction kinetics in a direct binding assay when the nanoparticles are sensitized through PAMAM G 1.5 to G4.5. A proper fit was appl ied to the plot, based on which Kon and Koff rates were determined.
Figure 11, shows the comparison in binding rate constants (k = konc+k0ffj offset corrected) obtained directly from the fit as wel l as the limiting val ue of relative fl uorescence intensity (//,m,rer loffset) at long time (over 600 min for CRP bind ing assays described in the present appl ication). The 'on' rate, konc, increased with increasing generation num ber of the dend rimer, consistent with the expected increase in the active surface area. The effect sign ificantly outweighed the effect of decreased diffusion coefficient of the na noparticle relative to that of the dye-cou ple anti body
While there is a lot of work on the fuctionalisation of silica N P with a reactive organosilane15,33, optimization of the l inker chemistry has not been previously investigated.
Accord ingly, the effect of the linker on N P sta bility and the performance of N P la bel led molecules in a sandwich immunoassay was investigated. In particular, the LOD and sensitivity was determined for the detection of human IgG. The N P la bel results were compared against results from a dupl icate assay performed using the same capture antibody la bel led with organic fluorophore, Cy5.
For th is study, three monovalent bilinkers (glutaraldehyde, sulfo-SMCC and SIAB), and th ree multivalent linker molecules (dendrimers, generations zero, one and two) were selected for the bioconjugation of N P with an anti body. Multivalent dendrimers are attracting a great deal of interest in biomedical d iagnostics34"37, pharmacology/drug delivery38, cancer research39, and nanomedicine40, and their use in research is still una bated now, 30 years after their first reported synthesis by Vogtle41.
It has been found that multivalency of dendrimers leads to a higher efficiency in bioconjugation of detection antibodies and an improved assay performance. Such improvements are necessary for reducing costs in medical diagnostics, particularly where large n umbers of samples are tested. Near-infrared dye doped sil ica N Ps, in which the fluorescence has been optimized were chosen for experimentation (Experimental and theoretical studies of the optimisation of fluorescence from near-infrared dye-doped silica nanoparticles, Nooney Rl, McCahey CMN, Stranik O, Guevel XL, McDonagh C, MacCraith BD, ANALYTICAL AND BIOANAL YTICAL CHEMISTRY, Volume: 393, Issue: 4, Pages: 1143-1149, Published: FEB 2009). At near infrared wavelengths, there is low background interference from the fl uorescence of biological molecules, solvent, and su bstrates. Furthermore, whole blood has a weak a bsorption in the N I R region, thus reducing the need for whole-blood filtering for assays using whole blood. N I R l ight can also penetrate skin and tissue to several mill imetres ena bl ing fl uorescence detection in dermatological or in-vivo diagnostic devices.
The three bivalent l inkers used are shown in the top half of Figure 1. The structure and mechanism for attachment of the dendrimers is shown in the bottom half of Figure 1. Nanoparticles were characterized using transmission electron microscopy and dynamic Light Scattering.
It should be noted that the num ber of dendrimers that may be accommodated on the nanoparticle surface wil l depend on the num ber of free amine groups provided on the nanoparticle surface. Typically, a nanoparticle of the invention may comprise surface amine groups on in the range of approximately 7 to 13% of the surface, with the remainder of the surface comprising sta bilizing phosphate groups.
If gl utaraldehyde, which may be considered a bi-l inker having two potential l in king sites, binds with one of its aldehyde groups to al l of the free a mine groups present on the nanoparticle surface having 13% amine covered surface, on ly 13% of the surface is availa ble to bind to a target protein. On the other hand, if the generation 0 dendrimer binds through one of its seven linking sites to al l of the free amine groups present on the nanoparticle surface having 13%, there are stil l another six linking sites availa ble to bind protein. This means that for nanoparticles having the same number of free amino groups on the surface, the num ber of potential protein or target binding sites is su bstantial ly increased by use of a dendrimer l in ker. While it is assumed that the dendrimer binds to the nanoparticle by a single linking -COOH group, it is possible that the dendrimer cou ld bind to the nanoparticle surface by more than one carboxylic acid group. The multivalency of the dendrimer means that it is possi ble that each dendrimer may l ink to multiple amines on the surface of the N P, assuming that the amines are in close to each other.
It is assumed that steric effects would l imit the num ber of target antibod ies which are capa ble of binding to the dendrimer to a bout fou r per nanoparticle. The num ber wil l also depend on the size of the protein and wil l increase with increased generation of the dendrimer. The type of target is also an important factor, for example, the num ber will vary depending on the protein, it wil l be different for the whole anti body as for some anti body fragments such as Fa b and ScFv. Figure 12 is a schematic of fuctionalisation of a nanoparticle with the preferred chemistry of the invention, immobilisation of dendrimer on the functionalised nanoparticle surface and use of the thus formed label in a bioassay. As shown in (1) the nanoparticle surface may be functionalised using a mixed of derivatized silanes, for example, phosphate and amine derivatized silane. In this particular example, a ratio of 10:1 phosphate compound to amine compound is used. This forms the functionalised nanoparticle surface, which preferably comprises in the range 7 - 13% surface coverage of -N H2 groups. Schematic (2) shows the "sensing layer" of dendrimer immobilised onto the functionalised nanoparticle surface and the subsequent attachment to target proteins through activation of the dendrimer terminal -COOH groups with sulfo-N HS/EDC coupling. This type of activation preserves the negative charge on the dendrimer and is crucial for colloidal stability of the resulting nanoparticle-dendrimer label.
Experimental Section
Materials
Triton ® X-100 (trademark Union Carbide), n-hexanol (anhydrous, >99%), cyclohexane (anhydrous 99.5 %), ammonium hydroxide (28 % in H20 > 99.99 %), tetraethylorthosilica (TEOS, 99.99 %), aminopropyltrimethoxysilane (APTMS, 97 %), aminopropyltriethoxysilane (APTES, 99 %), 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (TH PM P, 42 wt % in water), triethylamine (>99 %) absolute ethanol, monobasic sodium phosphate, dibasic sodium phosphate, phosphate buffered saline (PBS, pH 7.4, 0.01 M), Tween ® 20 (trademark Uniqema), glutaraldehyde (25 wt % in water), sodium azide (99.99 %) and albumin from bovine serum (BSA, 98 %) were all purchased from Sigma Aldrich Ireland, sulfo-SMCC and sulfo-SIAB were purchased from Pierce Chemical Company, dendrimers generations 1 and 2 were purchased from Frontier Scientific U K and used without further purification. However, it will be appreciated that any homemade or commercially available dendrimers that are soluble in the same solvent as the nanoparticles. Dendrimer generation 0 was synthesized in two simple steps starting from cyanuric chloride as starting material and purified on silica gel chromatography column prior the use43. Polyclonal Cy5 conjugated goat antihuman IgG (2.5 mg/mL in PBS), polyclonal goat anti human IgG (5 mg / mL in PBS) and polyclonal human IgG (5 mg/mL in PBS) were purchased from Biomeda Corp, California, USA. Black 96 well plates used in the immunoassay were purchased from AGB Scientific Ireland. Deionised water (< 18 ΜΩ) was obtained from a Milli-Q system from Millipore Ireland.
The dye used in this work is 4,5-Benzo-l'-ethyl-3,3,3',3'-tetramethyl-l-(4- sulfobutyl)indodicarbocyanin-5'-acetic acid N-succinimidyl ester, or more commonly referred to as N I -664-N-succinimidyl ester (purchased from Sigma Aldrich). This dye has a quantum efficiency of 23 %, a molar absorptivity of 187,000 L mol"1 cm"1 and fluorescence excitation and emission wavelengths of 672 nm and 694 nm, respectively, in isopropanol30. This dye is usually not used in immunoassays because it is not sol u ble in water.
Synthesis of silica NIR-NPs
Firstly, 15.6 mg N I -664-N-succinimidyl ester was dissolved in 5 m L anhydrous n-hexanol. To this solution, 5.021 μί of pure APTES and 3 μί of triethylamine were added. The mixtu re was agitated for 24 hours to ensure conjugation of the N I R dye to the organosilane. Briefly, the microemulsion was formed by mixing cyclohexane oil phase ( 15 m L), n-hexanol co-solvent (3.256 m L) and Triton ® X-100 surfactant (3.788 g) in 30 m L plastic bottles. To form the microemulsion, 0.96 m L of deionised water was added and the solution stirred for five min utes. Fol lowing this, 0.2 m L of TEOS and 0.16m L of N H4OH were added to start the growth of the silica N Ps. After thirty minutes, 0.344 m L of the N I R-664-APTES conjugate was added. The reaction was stirred for 24 hrs, after which 0.1 ml TEOS was added with rapid stirring.
After 30 minutes, 0.08 mL of the organosilane, 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (TH PM P), (42 wt % in water) was added with stirring to prevent aggregation of the nanoparticles19. After a further 5 minutes, 0.02 m L of bioreactive organosilane, aminopropyltrimethoxysilane, (APTMS) was added to and the solution stirred for a further 24 hou rs. The APTMS has a free primary amine group for crosslinking to biomolecules. The N Ps were separated from the solution with the addition of excess a bsolute ethanol and centrifuged twice with ethanol and once with deionised water. Sonication was used between the washing steps to resuspend the N Ps. The N Ps were dispersed in deionised water, at 2.0 mg / mL and stored in the dark at 4°C.
Bioconjugation
All bioconjugation reactions were performed with the N I R-N P at 2mg/m L, linker concentration of 4 m M and with 268 μg of goat anti-human IgG. The conjugation protocols were optimized for each linker with respect to the nature of its reactive groups.
Monovalently Linked NIR NP Bioconjugates
Gl utaraldehyde (4 μιτιοΙ) was added into a 1 m L solution of N I R-N P (2mg/m L) a nd gently shaken for 1 hour. The excess of unreacted l in ker was removed by means of centrifugation (3x) and the precipitates were dissolved in 946 μί of 0. 1 M PBS buffer, pH=7.4. To this sol ution, 54 μί (268 μg) of goat anti-human IgG was added and the mixture was al lowed to shake for 4 hou rs. The excess of the unreacted protein was removed by repeated centrifugation (4x) and the final bioconjugation product was dissolved in 1 mL of 0.1 M PBS buffer, pH=7.4. Sodium azide ( Na N3) was further added into the mixture so that its final concentration reached 0.01%.
Multivalently linked NIR NP Bioconjugates The bioconjugation reaction using the heterofunctional linkers comprised of two steps. First, sulfo-SIAB or sulfo-SMCC (4μιτιοΙ) were allowed to react with 1 mL solution of NI R-N P (2mg/mL) in 0.1 M PBS buffer, pH=7.2, containing 5 mM of EDTA for 30 minutes, after which the excess of the linker was removed by centrifugation. Second, 2-lminothiolane HCI (16.8 nmol, Traut's reagent) was reacted with goat anti-human IgG (268 μg, 1.68 nmol) in 250 μί of 0.1 M PBS buffer, pH=8.0, containing 5 mM of EDTA for 1 hour, after which the excess of the unreacted iminothiolane was removed using Zeba desalting column (Pierce Chemical comp.).
Both fractions were subsequently mixed together in a reaction vessel for 4 hours, while keeping the total volume of the solution to 1 mL. The reaction mixture was then purified by repeated centrifugation (4x) and re-dissolved in 0.1 M PBS buffer, pH=7.4, 0.01% NaN3.
Dendrimer NIR NP Bioconjugates
The nanoparticles as described herein comprise surfaces modified with -N H2 groups. The - COOH groups of all three generations of the dendrimers were first activated with EDC/N HS (1- ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (N HS)) mixture in 0.1 M MES buffer, pH=6.3 before reacting with the protein, goat anti human IgG.
For example, GO (4μιτιοΙ, 7x -COOH) was dissolved in 0.5 mL of M ES buffer. To this solution, N HS (42 μιτιοΙ, 1.5 equiv. per one -COOH group) and EDC (168 μιτιοΙ, 6.0 equiv. per one -COOH group) was added, the final volume was adjusted to 1 mL with MES buffer and the reaction was allowed to proceed for 30 minutes. White precipitates that were formed were separated in centrifuge. The clear liquid phase containing the N HS activated dendrimer was then directly added into the NI R-N P (2mg/mL), while keeping the total volume to 1 mL. This mixture was let to react for 30 minutes; subsequently the excess of solution-free dendrimer was removed by centrifugation. The dendritic-modified N I R-N P were re-dissolved in 946 μί of MES buffer, pH=7.0 and detection antibody, goat anti-human IgG (135 μg, 54 μί from stock) was added. The reaction was allowed to shake for 4 hours, then 100 μί of M ES buffer pH=9.0 was added to convert the remaining NHS esters into carboxyls and the mixture was purified by centrifugation (4x). The N IR- N P - IgG bioconjugate was re-dissolved in 0.1 M PBS buffer, pH=7.4, with 0.01 % NaN3.
All reactions in which N I R-N Ps were used were performed under reduced light (reaction vessels wrapped up in aluminium foil) to prevent photo-bleaching and all final samples were stored in dark at 4°C overnight before they were used in the immunoassay.
Fluorescence linked immunosorbent assay (FLISA)
A sandwich assay format was used to test the performance of the NPs compared to that of a single dye label. In the first step 100 μί of capture antibody, polyclonal goat anti human IgG, at 5 μg / mL was added to each well of a standard 96 well microplate. The plate was then incubated overnight at 4°C. To remove any non-adsorbed antibody the plate was rinsed three times with PBS and three times with PBS/0.05 wt % Tween ®. To prevent non-specific adsorption, 200 μί of 1 wt % BSA in PBS was added to each well and the plate incubated at 37 °C for 1 hour. The rinse cycle was then repeated. Following this, 100 μί aliquots of target analyte, antigen human IgG in 0.1 wt % BSA were added in a series of dilutions from 10,000 ng / mL to 0.1 ng / mL to each well and the plate incubated at 37 °C for 1 hour. The rinse cycle was repeated to remove any non-specifically bound human IgG. Finally, 100 μί aliquots of polyclonal goat anti-human IgG conjugated N Ps at 0.2 mg / mL were added to each well and the plate incubated for a further hour at 37 °C in the dark. Prior to analysis the plate was rinsed one more time. To compare the assay with a standard organic dye, the NP label was replaced with Cy5-conjugated goat antihuman IgG label at a concentration of 0.025 mg / mL.
The fluorescence signal including standard deviation at each concentration of human IgG was determined from seven replicate experiments. The complete assay was fitted using a standard sigmoidal logistics fit (see below, left hand side) and the low concentrations of human IgG were fitted using Ik afn allometric power function (see below, right hand side).
Figure imgf000031_0001
where, Fc is the fluorescence at concentration, C, F0 is the background fluorescence, Fmax is the maximum fluorescence, P is the power, and C0 is the point of inflection. For the allometric function, additional random variables, k, I, and m are required.
The LOD of human IgG was defined as the concentration corresponding to a fluorescence signal 3 times the standard deviation of the background signal. The sensitivity of the assay (change in fluorescence signal with concentration of human IgG) was determined at a range of concentrations through differentiation of the allometric power function. Previous work had indicated that the sensitivity of an assay is given without relating it to the concentration at which the sensitivity was measured. This can be misleading, since sensitivity does not change linearly with changing concentration.
Instrumentation
TEM micrographs were obtained using a Hitachi 7000 Transmission Electron Microscope operated at 100 kV. Images were captured digitally using a Megaview 2 CCD camera. Specimens were prepared by dropping aqueous solutions of the N Ps onto a formvar carbon coated copper grid. Fluorescence measurements were performed on a Safire (Tecan) microplate reader. For N I - 664-N-succinimidyl ester-doped N Ps, the excitation and emission wavelengths were set at 672 nm and 700 nm, respectively. Dynamic light scattering (DLS) measurements were performed on a Zetasizer from Malvern instruments to yield values of zeta potential (ξ) for the N Ps.
Results and Discussion
As seen in Figure 3, the monovalent linkers, glutaraldehyde and sulfo-SMCC have destabilizing effect on the N P with the tendency to form aggregates. Sulfo-SMCC shows two different populations, the first transition corresponds to the formation of nanoparticle trimers or tetramers, with an average diameter of 275 nm, and the second corresponds to larger aggregates with polydispersed characteristics.
On the contrary, NP modified with the multivalent dendrimers Gl and G2 show only a small increase in particle diameter, increasing the zeta potential from -29.8 to -47.9 and -46.0 for Gl and G2 respectively. The increased colloidal stability of dendrimer coated N Ps is due to the significant increase in the zeta potential on addition of the linker. This is because the N HS ester activated groups on the dendrimer are susceptible to hydrolysis, which leads to the formation of carboxylic groups that are charged at physiological pH. Therefore, the electrostatic charge on the NP is increased and colloidal stability is maintained.
Immobilization of a model protein, Green Fluorescent Protein (GFP), on the modified NP surface seemed to have little effect on the zeta potential of the NP modified with the Gl and G2, while the changes in case of Glu and sulfo-SMCC were more dramatic. The GFP attaches to the dendrimers. It was a surprising result to observe that the addition of the GFP did not detrimentally affect the zeta potential. It is therefore possible that there remains a large number of carboxyl groups on the surface to keep the electrostatic repulsion at a high level. This experiment was carried out to determine how much antibody would bind to the N Ps. The GFP is used as a substitute for a real antibody. From the fluorescence of the GFP the number of GFPs that are bound to the surface of our N P can be determined. The bioconjugation of the GFP on the dendrimer-modified surface was performed via a well-known technique for the conjugation of carboxyl (on the dendrimer) to amine groups (on the GFP) in peptides and proteins. The reaction between carboxylic acids and amines to form stable amides is commonly facilitated by an addition of a carbodiimide, such as l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC). However, EDC itself is not particularly efficient in linking because in the first step it forms an unstable O-acylisourea ester that is prone to fast hydrolysis and regeneration of the carboxyl. The carboxylic acids of the dendrimers were therefore activated in the presence of a mixture of N- hydroxysuccinimide (N HS) and a dehydrating agent, EDC. N HS/EDC mixture is commonly used to convert -COOH groups to semi-sta ble amine reactive N HS-esters. The availability and the lifetime of the NHS esters on the dendritic linker will determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labelled. Therefore, we decided to explore on the effect of some of the variables present in the EDC/N HS activation step such as the pH of the solution, the reaction time needed for the COOH activation, the molar excess of NHS per COOH group and the ratio of the EDC/NHS co-reactants.
It is noteworthy to mention that at these conditions, the optimum balance between the rate of aminolysis and the hydrolysis of the dendritic N-succinimidyl esters was observed. This turned out to be very important for maintaining high activity and specificity of the labelled antibody towards the antigen in the immunoassay.
After much experimenting with various parameters, the optimal conditions have been found and are as follows:
■ reaction medium - 0.1 M MES buffer, pH = 6.0;
■ activation time - 30 minutes;
■ two fold molar excess of NHS reagent per COOH group; and
■ 4: 1 ratio of EDC:N HS (See Figure 4, in particular).
Another very important parameter in the bioconjugation protocol is the concentration of the biomolecule that will comprise a monolayer on the NP surface. This amount is usually estimated and depends upon factors such as the molecular weight of the protein, its relative affinity for the particle and the particle size42. While the amount of protein required to achieve surface saturation can be calculated, the actual protein concentration in the reaction may be substantially higher than the "monolayer = 1 equivalent of protein" value. The optimal value is usually given by the coupling efficiency, concentration of the target molecules and the amount of the non-specific binding and is usually determined experimentally. Therefore, the effect of the concentration of the protein on the reaction yield was investigated. The coupling efficiency of two linkers, sulfo-SMCC and G2 in the bioconjugation reaction using a model protein (GFP) was compared. The reactions were carried out at two different concentrations of the GFP. First reaction was run with the amount of GFP representing 1.5 equivalent of the theoretical value necessary for surface saturation, and second reaction was performed using 3.0 equivalents. Figure 5 shows the effect of the increased concentration of GFP from 1.5 to 3.0 equivalents on the conjugation efficiency. The normalized fluorescence signal for sulfo-SMCC has nearly doubled at the higher GFP concentration (3.0) when compared to the lower GFP concentration (1.5). Nevertheless, it was still significantly lower than the signal observed for the multivalent G2 linker, and that applies even for the case when the G2 sample contained only 1.5 equivalents of GFP. It is also noteworthy that the differences between the conjugation efficiency (number of protein units (GFP) bound per nanoparticle) of the two G2 samples were very smal l. It suggests that the multivalency of the dendrimer is a significant factor responsi ble for the improvement in the reaction yields of conjugate, even at lower protein concentration^ Th is is very important, particularly when considering the cost of the bioorganic material that is used in bioconjugation reactions.
Fluorescence linked I mmunosorbent assay
To test the performance of the dye-doped silica N Ps, a standard fl uorescence l inked immunosorbent assay for the detection of human IgG was carried out. A sandwich assay format was used, in which the capture and detection antibod ies were polyclonal goat anti human IgG. Six different N P assays where performed using each of the mono- or multi- valent bilinkers. A further assay was performed using the same anti body la belled with the organ ic fluorophore Cy5. As seen in Figure 6, al l assays showed standard sigmoidal behaviour, excl uding N Ps prepared using G2 dendrimer, where it can be seen that the signal did not saturate at the highest concentrations. Saturation was not achieved since, at the time of the assay, sufficient dend rimer material was not availa ble to prepare the dendrimer standards at the highest concentrations. It is expected that when saturated standard solutions are used, the signal behaviou r wil l be sigmoidal . The normalized fluorescence ratio, at the h ighest concentration of human IgG, (Fmax/F0) for multivalent N P la bels and the organic Cy5 la bel was significantly greater than the fl uorescence achieved from la bels prepared using monovalent l inkers ( Figure 6 and Figure 7). Therefore, the n umber of N P la bels bou nd to human IgG is much greater than the number bound non-specifically. Fmax is the total nanoparticle signal (from specifically bound N Ps and non specifical ly bou nd N Ps), Fo is the signal from non specifica lly bound N Ps. The normal ized ratio is Fmax divided by Fo. If this ratio is high then this indicates that there is a lot more specifically bound sample than non specifical ly. Th is is indicative of a good assay.
As seen in Ta ble 1, all the multivalent la bels and the Cy5 la bel showed lower LO D than the monovalent linkers. Moreover, the LO D for G l dendrimer was greater than an order of magnitude lower than that for the Cy5 la bel . Therefore, multivalent N P are sensing lower concentrations of antigen human IgG and must be bound more strongly to the antigen coated surface on the plate. If lower concentrations of target are sensed, then the forces of interaction between the detection anti body coated N P and the antigen must be stronger. If binding is o bserved at lower concentrations, then the free energy is greater, indicating a stronger reaction between the nanoparticle and the target antigen.
From the work presented herein on the bioconjugation of G FP and other studies, it is l ikely the multivalent N Ps are coated with several detection antibod ies. This would increase the num ber of binding sites per la bel, increasing the reactivity of the la bel . Furthermore, because the footprint of a single N P la bel is significantly larger tha n the diameter of antigen, it may be possi ble for two antigens to bind to a single NP at the same time. The LOD for the dendrimeric NPs compares favorably with published data on the detection of biotinylated hlgG using avidin labelled silica NPs, where an LOD of 1.9 mg / mL was observed26.
For all three labels prepared using dendrimeric bi-linkers the assay showed good sensitivity at low concentrations of human IgG, with Gl showing the highest response (see Figure 8). For NP labels prepared using the monovalent bilinker and the Cy5 label no sensitivity was observed at concentrations of human IgG below 1 ng / mL. At higher concentrations all samples showed reasonable sensitivity.
Successful detection devices for biomedical diagnostics require high sensitivity and low LOD. Moreover, a device for point-of-care diagnostics, must be both inexpensive and reliable under a variety of experimental conditions. The data presented in this paper focused on fine- tuning the bioconjugation protocol in the labelling of biologically relevant material with NPs. We employed two types of linkers: monovalent linkers, glutaraldehyde; sulfo-SMCC; and sulfo-SIAB, dendrimers linker, in particular three generations of dendrimers with multivalent carboxylic functionality. The NP labels were compared against a commercial fluorophore label for the detection of human IgG. The NPs prepared using dendrimer generation 1 showed a significantly lower LOD and higher sensitivity than the commercial label. We believe the multivalency of the dendrimers is one of the most significant factors behind the increase in the detection sensitivity. As revealed in this work, dendrimers have a positive effect on NP stability and aggregation. Moreover, they are more reactive with biological materials, whilst maintaining activity and specificity to the analyte of interest. These properties are required for managing cost, in the design and implementation of biomedical devices, where expensive biological materials are necessary. Dendrimers are structurally appealing molecules that have already shown their high potential in the surface science of bioassay devices and this study confirmed that they are emerging as a powerful tool for the labelling of biomolecules in biomedical diagnostics as well.
It is shown how performance of an assay can be significantly improved when using multifunctional linkers such as dendrimers in the bioconjugation protocols. We demonstrated that with Newkome type dendrimers, Gen 1, the LOD was as low as 0.31 ng/mL. Nonetheless, the percentage of the NP surface area covered with the conjugated protein is still relatively low.
The conjugation protocol was further optimized and the surface coverage improved by using different type of dendrimers, polyamidoamine (PAMAM) dendrimers. PAMAM are the most common class of dendrimers suitable for many materials science and biotechnology applications. They are inexpensive, well soluble in aqueous media and come readily available in a range of generations. As seen on Figure 9, PAMAM dendrimers can increase the reaction yield and surface coverage by more than 20 times when compared to Newkome type dendrimers. It is believed that the protein coverage of the dye-doped nanoparticles is crucial element when designing point-of- care device as the key step in such a device is the binding rate of the detection antibodies. We hypothesize that the higher is the density of the detection antibody/protein on the N P surface, the faster is the kinetic response. This is very difficult to achieve with antibodies labelled with a single dye.
Preliminary data from a direct binding C P assay demonstrate the clear advantage of using higher generation of PAMAM dendrimer. generation 4.5 PAMAM has a binding rate nearly two times higher than its generation 1.5 PAMAM counterpart (Figure 10).
A point-of-care device requires low limit of detection, high sensitivity and ease of fabrication. But it also requires fast (usually within minutes) and precise detection, with a minimal error caused by a personnel manipulation. The use of dendrimers in the conjugation protocols seems to be a robust and reasonable way to prepare labelled antibodies necessary for ELISA type fluorescence assays with short response times and high sensitivity.
Conclusions
The biomolecular nanoparticle labels of the invention comprising nanoparticles having activatable carboxylate (-COOH) terminated dendrimers offers several distinct advantages over conventional bi- functional linkers and include,
(i) increased efficiency of conjugation reaction due to the multivalency of the dendrimer linker;
(ii) direct immobilization of biomolecules (proteins, DNA etc.) onto the dendrimer as the activatable - COOH groups can be directly activated using EDC and NHS/sulfo-NHS while maintaining the negative charge of the NPs during the conjugation reaction;
(iii) maintenance of the negative charge of the NP, allows the conjugation to biomolecules to be carried out in a monodispersed sample, thus increasing the efficiency of conjugation and increasing the overall population of 'antibody-sensitized-NP';
(iv) provides for formation of a flexible, 'soft' sensing layer that swells upon contact with water on the surface of the NPs and allows for efficient reaction between the NPs and antibodies;
(v) direct immobilization of proteins, for example, antibodies means the proteins are immobilized in their most active state and they retain their activity. Importantly, this applies not only to whole antibody but also to ScFV and Fab fragments;
(vi) By varying the generation of the for example, -COOH PAMAM dendrimer, a precise control over 'active surface area' of the nanoparticle is achieved. Lower dendrimer generations yield lower surface area (and vice versa) which can be loaded with target without significantly sacrificing the colloidal stability of the sample. The skilled person will appreciate that a number of these advantages also apply to -NH2 terminated dendrimers.
Thus, the labels of the invention present an opportunity for improvements of sensitivity in immunoassays through the use of dendrimers as antibody coupling agents for sensitisation of fluorescent nanoparticles.
Nanoparticles are useful as labels for antibodies in immunoassay procedures. As stated earlier, they can however suffer from some disadvantages including particle aggregation and nonspecific binding to the capture surface. Another issue in the use of nanoparticles as labels is the fraction of the antibody that is coupled to the particle that is in fact active for the reaction with antigen. This fraction can be rather small, which in turn can lead to diminished sensitivity and worsened non-specific binding.
One important element that affects the activity of the bound antibody and the non-specific aggregation and surface binding of particles is the means used for attachment of the antibodies. We have demonstrated significant improvements in the antigen-recognition activity of the sensitised particles, that correlates directly with the generation number of the dendrimer used for coupling.
We have also demonstrated that the use of dendrimers as the coupling agents suppresses non-specific binding effects to less than the level exhibited by a molecularly-labelled antibody. The net effect is a significantly improved signal/background.
The capture reaction, of particles onto the capture surface, can be written P + S - PS with forward rate constant kon and reverse rate constant koff. Here, S denotes an active site on the capture surface. If the particle is bound to the surface by a single antibody-antigen interaction, then k0ff will simply be the dissociation rate constant for the antigen-antibody complex. The association rate, kon, will be determined by the probability of a reactive collision between a particle and an un-occupied reactive site on the surface. Particles are in a continuous state of collision with the surface, at a rate determined by the diffusion coefficient of the particles and by their concentration. A reactive collision, leading to coupling of the particle to the surface, would occur when a reactive part of the surface of the particle (an antibody, oriented with the binding site exposed) collides with a reactive part of the surface (an antigen, oriented with the epitope exposed).
In the absence of any forces favouring a particular orientation of the particle with respect to the surface during approach and collision, the probability that the collision will be with an active site on the particle surface will simply be the fraction of the particle surface area that is covered by active antibody. As the particle concentration on the surface builds up, then the particles not only occupy sites but also physically block the surface area, on account of their size. So, the probability that the collision will be with an active site on the capture surface will simply be the fraction of the total surface that is unblocked by bound particles, times the fraction of this available capture surface area that is covered by active antigen, times a coverage-dependent factor that expresses the requirement that a particle requires a space whose smallest dimension is at least as large as the particle diameter, in order that the particle can fit into the space. This statement assumes that the approach and collision of particles is unaffected by the presence of previously bound particles except insofar as these occupy surface area. If koff is sufficiently low, then the requirement that particles can fit into the spaces available leads to a limiting coverage - the 'jamming limit' - which is approximately 53% of the total surface area for random sequential irreversible absorption of objects of circular cross-section.
If the coverage of the capture surface by bound particles is sufficiently low, then the effects of particles jamming the surface can be ignored, and the increase of particle coverage, and hence of surface-bound fluorescence, will follow a first-order rate equation:
Figure imgf000038_0001
Where / denotes the bound fluorescence intensity at time, t, with hypothetical value lmax if the surface were fully covered with fluorophore, and c denotes the particle concentration in the solution. The capture rate constant, kon can be written where kD is the diffusion-limit rate constant for particle consumption by the surface, dependent on the particle radius and the viscosity of the medium, e is the reaction efficiency for a reactive collision, qs is the fraction of the capture surface that is active for the capture reaction, in this case dependent on the surface coverage of adsorbed antigen, and qP is the active fraction of the particle surface, in this case dependent on the surface coverage of active antibody attached to the particle.
The objective of surface fuctionalisation with dendrimer is to increase qP. The exponential increase of average cluster volume with time in the reaction-limited aggregation of antibody- sensitised particles in the presence of the multi-valent antigen can similarly be described in terms of the probability of capture of antigen onto the surface of a particle or onto a particle at the outside of a cluster times the probability that this site will collide with an active site on another particle or cluster. Thus the rate constant for average cluster volume increase should increase approximately as (qP)2 for a given initial concentration of particles and antigen.
Figure 10 shows the fit of the bound fluorescence intensity to equation 1, with the addition of a fixed offset that we take to represent a non-specific adsorption of the sensitised particles. I n Figu re 11 we present the reaction parameters for the dend rimer-activated nanoparticles relative to those observed for an assay performed using anti body coupled directly to the fluorescent dye rather than to the nanoparticles loaded with fluorescent dye. The l imiting value of relative fl uorescence intensity at long time (over 600 min for C P binding assays described in the present appl ication) is — I0ff∞t} = f f— — I , where F is a scal ing factor.
The rate constant k = konc+koff is o btained directly from the fit. Th us the values of konc scaled relative to one another can simply be obtained from the relative values of the product k(liim,rerl offset)- The similarity of the val ues of (//,m,re/-/o//set) scaled relative to the dye-coupled anti body with those of konc similarly scaled implies that koff » konc. The variation of the rate constant, k, is thus that of the off rate, koff. as expected, this was within the experimental uncertainty the same for the dye-la belled and particle-la bel led antibodies, and not affected by the nature of the dendrimer cou pling compound (Figure 11). The 'on' rate, konc, increased with increasing generation num ber of the dendrimer, consistent with the expected increase in the active surface area. The effect significantly outweighed the effect of decreased diffusion coefficient of the nanoparticle relative to that of the dye-couple antibody.
The other important characteristic of the la bels to consider for use in an assay is the ratio of signal to background : in this case, the ratio of the equil ibrium fl uorescence (offset corrected) to the non-specific offset. Again, a significant advantage is noted for the dendrimer-sensitised nanoparticle la bels. The offset was hardly affected, indeed may have been reduced. The signal was significantly enhanced so the relative val ue of signa l/non-specific background was increased by a factor of ~3 as a consequence of the use of the dendrimer-sensitised nanoparticles as the la bel .
All in all, these effects lead to the provision of superior la bels for use in assays capa ble of detecting target analyte to the ultra low LOD level.
The words "comprises/comprising" and the words "having/incl uding" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not precl ude the presence or addition of one or more other features, integers, steps, components or groups thereof.
It is appreciated that certain featu res of the invention, wh ich are, for clarity, descri bed in the context of separate embodiments, may also be provided in com bination in a single em bodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single em bodiment, may also be provided separately or in any suita ble su bcombination. It will be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalised examples only, and that other arrangements and methods capable of reproducing the invention are possible and are embraced by the present invention.
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(43) Intermediate 1: To a pressure vessel containing 100 mL of CH2CI2:MeOH (1:1), 2-Amino-2- (hydroxymethyl)-l,3-propanediol (3.9 g, 33 mmol) and Hunig's base (8.4 g, 65 mmol) were added. The mixture was cooled in an ice bath before addition of cyanuric chloride (3 g, 16 mmol). The reaction was then al lowed to warm up to room temperature and stirred for 24 hours before 4-piperidine carboxylic acid (8.4 g, 65.2 mmol) and N H4OH (5 m L) were added. The reaction mixture was then heated up to 60°C and stirred overnight. Upon cooling, the precipitates were filtered and the solvent was removed by evaporation. The residue was re- dissolved in water (20 mL) and acidified to pH = 4-5. Precipitates were formed, filtered off and washed extensively with water to give off-white powder (3.44 g, 47 % yield). This intermediate was used in the second step without further purification.
Dend rimer GO: Intermediate 1 (0.5 g, 1.1 mmol ) and diglycolic anhydride (3 g, 16 mmol ) were placed in a sealed vessel and irradiated in a CEM microwave at 300 W, 120°C for 5 minutes. U pon cool ing, precipitates were formed, the whole mixture was dissolved in water and the pH was adjusted to 4-5. The sol ution was concentrated and the residue was purified on silica gel ch romatography column with MeO H/Ch^C^ (5:95) as an eluent to yield oily product (440 mg, 35% yield).

Claims

Claims
1. A label comprising:
a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable -COOH group for conjugating a target analyte to the functionalised nanoparticle,
wherein the dendrimer has a stabilizing effect on the zeta potential at the nanoparticle surface.
2. A label according to claim 1 wherein the nanoparticle surface fuctionalisation comprises a first ionisable functional group which is inert to a target analyte and a second ionisable group functional group for immobilising at least one dendrimer onto the nanoparticle surface, wherein the first and second ionisable functional groups have a different electrostatic charge and the second ionisable functional group is present on the nanoparticle surface in an amount sufficient to prevent nanoparticle aggregation in a colloidal solution.
3. A label according to claim 2 wherein the first ionisable function group is present on the nanoparticle surface in a range of about 87% to 93% of the total nanoparticle surface area.
4. A label according to claim 2 or 3 wherein the second ionisable function group is present on the nanoparticle surface in a range of about 7% to 13% of the total nanoparticle surface area.
5. A label according to claim 3 or 4 wherein the first ionisable functional group is a phosphate group.
6. A label according to claim 3 to 5 wherein the second ionisable functional group is a N H2 group.
7. A label according to any preceeding claim wherein the dendrimer is a Newkome whole generation dendrimer selected from the group consisting of: a generation 0 to generation 10.
8. A label according to any one of claims 1 to 6 wherein the dendrimer is a PAMAM half generation dendrimer selected from the group consisting of: Generation 0.5 dendrimer, a Generation 1.5 dendrimer, a Generation 2.5 dendrimer, a Generation 3.5 dendrimer, a Generation 4.5 dendrimer, a Generation 5.5 dendrimer or a Generation 6.5 dendrimer.
9. A label according to any preceeding claim wherein the dendrimer is a Generation 4.5 dendrimer.
10. A la bel according to any one of claims 1 to 6 or 8 wherein the dendrimer is a Generation 2.5 dendrimer.
11. A la bel according to a ny preceding claim wherein dendrimer functional ised nanoparticle may be conjugated to a detection molecule which detects a target analyte.
12. A la bel accord ing to claim 9 wherein the detection molecule is selective for a articular target analyte.
13. A la bel according to any preceeding wherein the nanoparticle is a sil ica nanoparticle or a metal oxide nanoparticle.
14. A la bel according to any preceeding claim wherein the nanoparticle is at least one of dye-doped, magnetic, sil ica or metal doped .
15. A la bel according to any preceeding claim wherein the nanoparticle is a hybrid nanoparticle comprised of a hybrid material and include, noble metal, quantum dot or magnetite doped silica nanoparticles.
16. A la bel according to claim 14 wherein the dye-doped nanoparticle is fluorescence optimized for near infrared electromagnetic radiation.
17. A la bel according to any preceeding claim wherein the nanoparticle has a diameter in the range of a bout 10 nm to a bout 1000 nanometres.
18. A la bel according to any preceeding claim wherein the nanoparticle has a surface layer comprising an orga nosilane with one or more functional groups, for example, amines, thiols, phosphates, epoxy, carboxyl, cyano, isothiolcyanate, iodoacetamide, azido, NHS-ester.
19. A la bel according to claim 18 wherein the organosilane comprises an organo group comprising at least one of thiol, amine or carboxylate, phosphate, epoxide or isothiocynate functional groups.
20. A la bel according to any one of claims 18 to 19, wherein the surface layer further comprises at least one of a generation 0 to a generation 10 dendrimer and half generations thereof.
21. A la bel according to any preceeding claim wherein the surface layer has a th ickness in the range of a bout 1 nm to a bout 100 nanometres.
22. A la bel according to any preceeding claim wherein the nanoparticle surface is fu rther functionalised with at least one of an activata ble biochemical group including thiol, amine or carboxylate, phosphate, epoxide or isothiocynate.
23. A la bel according to any preceeding claim wherein the nanoparticle is further functionalised with at least one of a protein, a nucleic acid, a molecular la bel, a molecular tag, a fl uorophore or a nanoparticle.
24. A la bel according to any preceeding claim wherein the ta rget analyte is a cell, a pathogen, a protein, a molecular la bel, a molecular tag, a nucleic acid, a detection molecu le or a secondary analyte selective for a further analyte species.
25. A la bel according to any one of claims 23 or 24 wherein the molecular tag is bioton or streptavadin.
26. A la bel according to any one of claims 23 to 25 wherein the molecu lar la bel is a chemosensor or a protein fluorescent probe.
27. A la bel accord ing to any one of claim 23 to 26 wherein the protein is green fluorescent protein.
28. A la bel according claim 24 wherein the target analyte, the detection molecule or the secondary analyte is protein, incl uding a monoclonal anti body, a polyclonal, recom binant anti body, antibody fragment or an antigen.
29. A la bel comprising a nanoparticle wherein the nanoparticle surface is functional ised to comprise at least one dendrimer having at least one activata ble functional grou p for conjugating a detection molecule selective for a corresponding target analyte, wherein the dendrimer increases the zeta potential of the nanoparticle.
30. A la bel according to claim 29 wherein the detection molecule is a monoclonal anti body, a polyclonal anti body or an antigen.
31. A la bel according to any one of claims 24 to 30 wherein the anti body is goat antihu man IgG.
32. A la bel according to any one of claims 24 to 31 wherein the antigen is polyclona l human IgG
33. Use of a la bel accord ing to any preceeding claim in an immunoassay.
34. Use of a la bel according to claim 32 wherein the immunoassay is an immunosorbent assay, such as an E LISA, a FLISA, a fluorescent enzyme-l inked immunosorbent assay, a sandwich enzyme-l inked immunosorbent assay, a sandwich fl uorescent enzyme-l in ked immunosorbent assay, a competitive enzyme-l inked immunosorbent assay or a direct binding assay.
35. Use of a la bel according to any one of claims 1 to 34 as a tool for biological discovery and/or biomedical detection, in medical imaging and/or therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.
36. Use of a label according to any one of claims 1 to 34 in a dermatological or in vivo diagnostic device.
37. A method of preparing a nanoparticle label according to any one of claims 1 to 32 comprising the steps of:
(i) activating at least one functional group on a provided nanoparticle to provide at least one activated functional group having enhanced reactivity; and
(ii) condensing the at least one activated functional group with a functional group of a provided dendrimer to link the dendrimer to the nanoparticle; and optionally,
(iii) quenching the at least one activated function group.
38. A method according to claim 37 wherein the label is reactivated before use.
39. A method according to any one of claims 37 or 38 further comprising the step of attaching at least one detection molecule to the activated label to form a label.
40. A method according to claim 39, wherein the detection molecule is selective for a target analyte.
41. A method of detecting a target analyte in a test substance using the label according to any one of claims 1 to 32 comprising the steps of:
(i) activating at least one of the activatable functional groups on the dendrimer to form an activated label; and
(ii) exposing the activated label to the test substance comprising the target analyte; and
(iii) analysing the test substance for bound label.
42. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of:
(i) immobilising a target analyte onto a support;
(ii) washing the support to remove unbound target analyte;
(iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
(iv) washing the support to remove unbound activated label;
(v) analyzing for bound target analyte.
43. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of:
i) immobilising a target analyte onto a support;
ii) washing the support to remove unbound target analyte;
iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
iv) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule;
vi) exposing the activated label to the support so that detection molecule binds to the secondary analyte molecule;
vii) washing the support to remove unbound activated label; and
viii) analyzing for bound label.
44. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of:
i) immobilising a target analyte onto a support;
ii) washing the support to remove unbound target analyte;
iii) exposing the support to a secondary analyte molecule which binds to the target analyte;
iv) washing the support to remove any unbound secondary analyte molecule v) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte;
vi) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule;
vii) washing the support to remove unbound activated label;
viii) analyzing for bound label.
45. A method according to any one of claims 42 to 44, wherein the first step of immobilising the target analyte onto the support comprises:
(i) immobilising a capture molecule selective for the target analyte onto the support;
(ii) washing the support to remove unbound capture molecule; and (iii) exposing the support to the target analyte to immobilise the target analyte onto the immo bil ised capture molecule.
46. A method according to any one of claims 42 to 45 wherein target analyte is extracted from a test su bstance, including blood, tissue or a blood derivative or a tissue derivative.
47. A method according to any one of claims 37 to 46 wherein the activating step involves treating the nanoparticle la bel with N-hydroxysuccinimide ( N HS) or sulfo N- hydroxysuccinimide and a dehydrating agent.
48. A method of detecting an analyte according to claim 47 wherein the dehydrating agent is l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ( EDC).
49. A method of detecting an analyte according to claims 48 wherein the nanoparticle la bel is activated by treating the nanoparticle la bel with N-hydroxysuccinimide ( N HS) or sulfo N-hydroxysuccinimide and a dehydrating agent l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ( EDC) in a M ES buffer having a concentration in the range a bout Im M to a bout 1M.
50. A method according to claim 49 wherein the concentration of the M ES buffer is 0.1M.
51. A method of detecting an analyte according to claims 49 wherein the buffer has a p H in the range a bout 3 to a bout 9.
52. A method according to claim 51 wherein the pH of the M ES buffer is pH = 6.
53. A method of detecting an analyte according to any one of claims 49 to 52 wherein the activation period is in the range a bout 10 seconds to a bout 30 minutes.
54. A method according to claim 53 wherein the activation period is about 2 hours.
55. A method of detecting an analyte accord ing to any one of claims 37 to 54 wherein the ratio of EDC: N HS is in the range a bout 1 :2 to a bout 4: 1.
56. A method of detecting an analyte according to any one of claims 49 to 56 wherein the N-hydroxysuccinimide ( N HS) or sulfo N-hydroxysuccinimide is present in a molar excess in the range of N HS reagent per activata ble group.
57. A methods according to claim 56 wherein the molar excess is 2: 1.
58. A la bel prepared according to the method as claimed in any one of claims 37 to 57 wherein the activated dendrimer surface modified nanoparticle comprises semi-sta ble amine reactive N HS-esters.
59. A method for preparing a label comprising the steps of:
(i) functionalising a nanoparticle surface with a first ionisa ble functional group which is inert to a target analyte and with a second ionisa ble fu nctional group for immobilising at least one dendrimer onto the nanoparticle surface, wherein the first and second ionisable functional groups have a different electrostatic charge and the second ionisable functional group is present on the nanoparticle surface in a amount sufficient to prevent nanoparticle aggregation in a colloidal solution;
(ii) immobilising a dendrimer onto the nanoparticle surface by reacting the second ionisable functional group with a suitable functional group on the dendrimer, the dendrimer having at least one activatable functional group for conjugating a target analyte and wherein the dendrimer is terminated with a third functional group of the same charge as a first ionisable functional group on the nanoparticle and wherein the third functional group is reactive towards the target analyte.
60. The method according to claim 59 wherein the activatable functional group of the dendrimer is a carboxylate.
61. The method according to claim 59 or 60 wherein the first ionisable functional group is phosphate.
62. The method according to claim 59 to 61 wherein the second ionisable functional group is -N H2.
63. The method according to claim 59 to 62 wherein the third functional group is -COOH.
64.
A label substantially as hereinbefore described with references to the accompanying drawings.
65. A label substantially as hereinbefore described with references to the accompanying examples.
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