WO2010110749A1 - Method of loading nucleic acids to noble metal surfaces - Google Patents

Abstract

The present invention provides a method of immobilizing a nucleic acid on a substrate having a noble metal surface. The method includes contacting the nucleic acid with the substrate, wherein the substrate comprises a non-ionic surfactant on its noble metal surface. The present invention also provides a method of detecting a target nucleic acid. The present invention also refers to a substrate obtained by the method and a kit comprising a substrate obtained by the method.

Description

METHOD OF LOADING NUCLEIC ACIDS TO NOBLE METAL SURFACES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application makes reference to and claims the benefit of priority of an application for "Facile Loading Of Single Stranded DNA On Gold Nanoparticles With Conformational Control" filed on March 25, 2009 with the Intellectual Property Office of Singapore, and there duly assigned serial number 200902068-6. The content of said application filed on March 25, 2009 is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

TECHNICAL FIELD

[0002] The present invention refers to the field of biochemistry and in particular to technologies concerning immobilization of biomolecules and detection of nucleic acids.

BACKGROUND

[0003] Detection of specific oligonucleotide sequences has important applications in medical research, clinical diagnosis, food and drug industry, and environmental monitoring.

[0004] Most assays identify specific nucleic acid sequences through hybridization of an immobilized nucleic acid probe to the target nucleic acid after the latter has been modified with a covalently linked label such as a fluorescent or radioactive tag. For example, gold nanoparticles covalently functionalized with DNA sequences to bind specific target DNA sequences for oligonucleotide sensing have also been used. Such functionalized nanoparticles are used as building blocks for nanoscale materials and as novel reagents for biosensing and gene regulation.

[0005] Despite efforts in this area of research, it remains a challenge to immobilize biomolecules, such as single-stranded DNA (ssDNA), facilely and controllably on gold surfaces. For example, ssDNA strands with thiol linkers have been used for attachment onto gold surfaces. Surface density of ssDNA immobilised on gold surfaces is dependent on ionic strength of the immobilization solution, as electrostatic repulsion force exists between ssDNA strands. [0006] Sodium chloride (NaCl) salt solutions have been used as immobilization solutions. However, when gold nanoparticles are used, the salt solution may destabilize the gold nanoparticles and cause the particles to aggregate irreversibly. For example, state of the art methods to prepare ssDNA-gold nanoparticle conjugates includes a salt aging process, in which sodium chloride solution is added gradually at 10-50 mM doses to a gold nanoparticle solution containing thiolated ssDNA. The solution was allowed to incubate for a couple of hours following each dose. Upon reaching a final sodium chloride concentration of 1.0 M, a further incubation time of one to two days was required. As a result, the whole salt aging process could take several days. Although some research groups have recently reported a reduction in preparation time of such ssDNA-gold conjugates to one day, the preparation procedure is still tedious as it was necessary to add salt solution gradually in a stepwise fashion.

[0007] Conformational control of surface-bound biomolecules, such as nucleic acids, is another challenge that exists. Studies by various research groups have indicated that non- specific adsorption of nucleic acids on gold surfaces is significant. One reason is because thiolated nucleic acid molecules interact with gold surfaces through their thiol groups, as well as their amine groups in nucleotide bases. The adsorption of nucleobases on gold surfaces may compromise functionality of the nucleic acid sensors. To optimize orientation of the surface-bound nucleic acids, mercaptohexanol (MCH) has been used to treat nucleic acid- modified gold surfaces. Mercaptohexanol molecules work to displace some of the non- specifically adsorbed nucleic acid molecules, which frees up the space originally occupied by non-specifically adsorbed nucleic acid molecules for thiolated nucleic acids to interact with the gold surface. However, in the case of nucleic acid-gold nanoparticle conjugates, excessive displacement of the non-specifically adsorbed nucleic acid by mercaptohexanol could result in destabilization of the gold nanoparticles. As such, precise control of mercaptohexanol concentration and reaction time is required. In addition, there is also addition process step of using ethyl acetate to remove excess mercaptohexanol from the solution.

[0008] It is therefore an object of the present invention to provide alternative methods to immobilize biomolecules on surfaces with properties that overcome at least some of the above described disadvantages.

SUMMARY OF THE INVENTION

[0009] In a first aspect the present invention provides a method of immobilizing a nucleic acid on a substrate having a noble metal surface. The method includes contacting the nucleic acid with the substrate, wherein the substrate comprises a non-ionic surfactant on its noble metal surface.

[0010] In a second aspect the invention provides a method of detecting a target nucleic acid. The method includes immobilizing a capture nucleic acid adapted to hybridize to the target nucleic acid on a noble metal surface of a substrate, wherein a no n- ionic surfactant is immobilized on the noble metal surface. In a further step, the method includes contacting a sample suspected to contain the target nucleic acid with the substrate.

[0011] In a third aspect the invention provides a substrate for detecting a target nucleic acid. The substrate comprises a noble metal surface. The substrate has immobilized on the noble metal surface, a non-ionic surfactant and a capture molecule adapted to hybridize to the target nucleic acid.

[0012] In a fourth aspect the invention provides a kit for detecting a target nucleic acid. The kit includes a substrate comprising a noble metal surface. The substrate has immobilized on the noble metal surface, a non-ionic surfactant and a capture molecule, wherein the capture molecule is adapted to hybridize to the target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The invention will be better understood with reference to the detailed description when considered in conjunction with the accompanying drawings. [0014] Fig. l(a) discloses the general principal of the method of immobilizing nucleic acid. As shown in Fig. l(a), a substrate having a noble metal surface, wherein the noble metal surface comprises a non-ionic surfactant, is contacted with a solution comprising nucleic acid molecules. Each nucleic acid molecule can comprise a chemical linker adapted to interact with the noble metal surface. After contacting, the nucleic acid molecules bind to the noble metal surface via the linker. Fig. l(b) shows an embodiment of the present invention, in which the substrate is a nanoparticle.

[0015] Fig. 2(a) shows the loading profile of thiolated ssDNA bearing a Tio spacer (Probe 1). Fig. 2(b) shows the loading profile of thiolated ssDNA bearing a T₂₀ spacer (Probe 2). Fig. 2(c) shows the loading profile of thiolated ssDNA bearing an iSplδ spacer (Probe 3). Fig. 2(d) shows the loading profile of thiolated ssDNA with iSplδ and T₂₀ spacer (Probe 4). All figures show that an increase of sodium chloride concentration led to a higher loading density of the ssDNA.

[0016] Fig. 3 shows a comparison graph of surface density of non-specifically adsorbed ssDNA (non-thiolated) on the gold nanoparticles. The chart shows a much lower level of non- specifically adsorbed ssDNA (non-thiolated) on FSN-stabilized gold nanoparticles compared to that of citrate-stabilized gold nanoparticles.

[0017] Fig. 4(a) is a graph showing the surface density of thiolated ssDNA (Probe 1) on 13 nm gold nanoparticle as a function of incubation time. Fig. 4(b) is a graph showing fluorescence evolution of the mixture solution of the ssDNA (Probe 1*) and the FSN- stabilized gold nanoparticles as a function of incubation time. It can be seen from both graphs that ssDN A layer formation reaches steady state after about two hours.

[0018] Fig. 5 is a graph showing fluorescence evolution of the mixture solution of an FAM- labelled non-thiolated ssDNA (Probe 1 '*) and a 13 nm FSN-stabilized gold nanoparticles as a function of incubation time. It can be seen from the graph that non-thiolated ssDNA cannot be immobilized through non-specific adsorption. [0019] Fig. 6 is a graph showing the hybridization efficiency as a function of surface density of thiolated ssDNA on 13nm gold nanoparticles for thiolated ssDNA bearing T₁₀ (Probe 1), T₂o (Probe 2), and iSpl8 (Probe 3) spacers. The graph shows that the hybridization efficiency is a function of the surface density of the probes. Hybridization behaviour was also found to be sensitive to spacer length of the thiolated ssDNA.

DETAILED DESCRIPTION OF THE INVENTION

[0020] In a first aspect the present invention provides a method of immobilizing a nucleic acid on a substrate having a noble metal surface. The method includes contacting the nucleic acid with the substrate, wherein the substrate comprises a non-ionic surfactant on its noble metal surface. [0021] The use of an adlayer of a non-ionic surfactant prevents nucleobases of nucleic acids from interacting with the noble metal surface, thereby suppressing non-specific adsorption of nucleic acid on noble metal surface. As interaction between the nucleobases and the noble metal surface is now inhibited by the layer of non-ionic surfactant, there is no slow reorganization of molecular configuration in the adsorption process. The nucleic acid molecules displace the adsorbed non-ionic surfactant molecules and are thus immobilized at an upright orientation. Using this method the control of nucleic acid conformation is realized. This control of nucleic acid conformation results in high hybridization efficiency, since most of the nucleic acid may be bound with an upright orientation and are available to hybridize with their target nucleic acids.

[0022] The term "nucleic acid" as used herein refers to any nucleic acid in any possible configuration, such as single stranded, double stranded or a combination thereof. Nucleic acids include for instance DNA molecules, RNA molecules, analogues of the DNA or RNA generated using nucleotide analogues or using nucleic acid chemistry, locked nucleic acid molecules (LNA), PNA molecules and tecto-RNA molecules (e.g. Liu, B., et al., J. Am. Chem. Soc. (2004) 126, 4076-4077). An LNA molecule has a modified RNA backbone with a methylene bridge between C4' and 02', which locks the furanose ring in a N-type configuration, providing the respective molecule with a higher duplex stability and nuclease resistance. Unlike a PNA molecule an LNA molecule has a charged backbone. DNA or RNA may be of genomic or synthetic origin and may be single or double stranded. Such nucleic acid can be e.g. mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, a copolymer of DNA and RNA, oligonucleotides, micro RNA having a length of between about 21 to 23 nucleotides etc. A respective nucleic acid may furthermore contain non-natural nucleotide analogues.

[0023] Many nucleotide analogues are known and can be present and/or used in the methods of the invention. A nucleotide analogue is a nucleotide containing a modification at for instance the base, sugar, or phosphate moieties. As an illustrative example, a substitution of 2'-OH residues of siRNA with 2'F, 2'O-Me or 2'H residues is known to improve the in vivo stability of the respective RNA. Modifications at the base moiety include natural and synthetic modifications of A, C, G, and T/U, different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl, and 2-aminoadenin-9-yl, as well as non-purine or non- pyrimidine nucleotide bases. Other nucleotide analogues serve as universal bases. Universal bases include 3-nitropyrrole and 5 -nitro indole. Universal bases are able to form a base pair with any other base. Base modifications often can be combined with for example a sugar modification, such as for instance 2'-O-methoxyethyl, e.g. to achieve unique properties such as increased duplex stability. [0024] The nucleic acid referred to herein can be immobilized on the surface for the detection of target nucleic acids in a solution. As such the nucleic acid immobilized on the surface can also be referred to as nucleic acid probe. In one embodiment, the nucleic acid probe is a nucleic acid with a substantially negative net charge. For example, a nucleic acid probe with a substantially negative net charge could comprise a single stranded DNA (ssDNA). In one embodiment, the nucleic acid probe is a nucleic acid with a substantially neutral net charge. For example, a nucleic acid probe with a substantially neutral net charge could comprise PNA A nucleic acid probe with a substantially neutral net charge could be made substantially negative through introduction of negative charges via inclusion of glutamic acid and/or aspartic acid group without affecting its ability to bind to the substrate having a noble metal surface.

[0025] The nucleic acid immobilized to the substrate having a noble metal surface can be fixed to the noble metal surface via an additional chemical linker attached to the nucleic acid. A chemical linker is a chemical group attached to the nucleic acid which is adapted to interact with the noble metal surface, i.e. in one embodiment the chemical linker would be arranged between nucleic acid and the noble metal surface. The chemical linker can bind to the nucleic acid and/or the noble metal surface via covalent bonding, ionic bonding or Van-der-Waals bonding. A chemical linker includes a functional group, such as a thiol group, an amino group, a hydro xyl group, or an epoxide group. Thiol groups are used most often to link nucleic acids to a noble metal surface. The usability of each group may depend on several conditions, such as the respective applications and the reaction conditions, and can be determined empirically.

[0026] The nucleic acid may additionally comprise a spacer. The term "spacer" as used herein refers to a chemical structure which links two components to each other. The spacer can be used to modify the distance between the noble metal surface and the nucleic acid. Furthermore, the spacer can be located between the chemical linker which is bound to the noble metal surface and the nucleic acid. Thus, a spacer is used to increase the distance between surface and nucleic acid. [0027] A spacer can also be used to minimize steric effects caused by the substrate. Examples of spacer can include, but are not limited to, a nucleic acid spacer, an alkane spacer, a polypeptide spacer, a nucleotide spacer, such as an adenosine spacer or a thymine spacer; a polyethylene glycol (PEG) spacer, phosphoramidite, or l',2'-dideoxyribose, and derivatives thereof. A polyethylene glycol spacer can have a length of at least two ethylene glycol molecules. Examples of a polyethylene glycol spacer can include triethylene glycol and 18- atom hexa-ethyleneglycol (iSpl8). Length of the spacer can be varied for specific applications. For example, thymine spacer can be in the form of thymine TlO, which is a 10 thymine base-long oligonucleotide or in its longer form, thymine T20, which is a 20 thymine base-long oligonucleotide. A spacer, such as a nucleotide spacer or a polyethylene glycol spacer, can have a length of between about 2 to 60 units for example. One unit corresponds to a specific repeating molecule in a spacer. For example, in a polyethyleneglycol spacer one unit would be one ethyleneglycol molecule. In a nucleotide spacer one unit would be one nucleotide.

[0028] The term "immobilized" as used herein refers to the state at which freedom of motion of the nucleic acid is prevented. The nucleic acid can be immobilized on a substrate via physical binding or chemical binding. For example, physical binding can take place due to attractive forces such as Van-der-Waals forces between the nucleic acid and noble metal surface. As a further example, physical binding can take place as a result of nucleic acid molecule being held in place on a surface due to steric effects. For chemical binding, this can be done via chemical linkages such as covalent bonding, for example thiol-noble metal bonding, between the nucleic acid and noble metal surface. Generally, the nucleic acid can also be immobilized on a surface directly without any chemical linker or spacer or any other group in between. In one embodiment, the nucleic acid can be immobilized on the surface with a chemical linker in between. In another embodiment, the nucleic acid can be immobilized on the surface with a chemical linker and a spacer in between.

[0029] Surfactants can be categorized according to the charge present in the hydrophilic portion of the molecule (after dissociation in aqueous solution). They can be classified as anionic, non-ionic, cationic and zwitterionic surfactants. In particular, many non-ionic surfactants comprise one or more chains or polymeric components having oxyalkylene (-O-R-) repeats units, wherein R has 2 to 6 carbon atoms. Representative non-ionic surfactants comprise block polymers of two or more different kinds of oxyalkylene repeat units. Examples of a non-ionic surfactant can include, but are not limited to fluorosurfactant, polyoxythylene based surfactant, octyl glucoside, sorbitan ester, hydrogenated surfactants, poloaxamers, alkyl poly(ethylene oxide), diethylene glycol monohexyl ether, polyvinyl alcohole (PVA), copolymers of poly(ethylene oxide) and poly(propylene oxide), hexaethylene glycol monohexadecyl ether, alkyl polyglucosides, digitonin, ethylene glycol monodecyl ether, cocamide MEA, cocamide DEA, cocamide TEA, fatty alcohols, or mixtures thereof. Examples of commercially available non-ionic surfactants include ZONYL^® series of non- ionic fluoropolymers available from Sigma-Aldrich, 3M™ Novec FC series of non-ionic fluoropolymers from 3M (St. Paul, MN.), Masurf^® FS series of non-ionic fluoropolymers from Mason Chemical Company (Arlington Heights, IL), TETRONIC^® series of tetrafiinctional block copolymers of propylene oxide, ethylene oxide, and ethylene diamine available from BASF Canada (Toronto, Ontario), the TERGITOL^® series of alkyl polyethylene oxides available from Union Carbide Co. (Houston, Texas), the BRIJ^® series of polyethoxylated alcohols and esters available from ICI Americas (Wilmington, Del.), the SURFYNOL^® series of acetylenic polyethylene oxides available from Air Products (Allentown, Pa.), or the TRITON^® series of alkyl phenyl polyethyleneoxides available from Rohm & Haas (Philadelphia, Pa.)

[0030] Fluorosurfactants are analogs of conventional hydrocarbon surfactants wherein a part or even all of the hydrogen atoms along the carbon molecular backbone have been replaced with fluorine atoms. They are characterized as being both anionic and nonionic surfactants, a well known example of which is perfluorooctanoic acid. Examples of non-ionic fluorosurfactants can include, but are not limited to, the ZONYL^® series of fluoropolymers including Zonyl^® FSA, FSP, FSE, UR, FSJ, FSO, FSO-IOO, FS-300, FSN, FSN-100 and TBS available from Sigma-Aldrich, 3M™ Novec FC-4434 from 3M (St. Paul, MN.), Maflon^® Lineplus PDM series from Maflon and Masurf^® FS- 1400, FS- 1900 and FS-2000 from Mason Chemical Company (Arlington Heights, IL).

[0031] In the ZONYL^® series of fluoropolymers, ZONYL^® FSO, ZONYL^® FSN, and ZONYL^® FS-300 are exemplary non- ionic fluorosurfactants that may be used in the present invention. These non-ionic fluorosurfactants are made up of a hydrophilic part comprising a polyethoxyethylene chain, and a hydrophobic part comprising a fluorocarbon chain. They have the general structure Rf-CH₂CH₂O(CH₂CH₂O)_xH, wherein Rf has the general formula F(CF₂CF₂)_y. For ZONYL^® FSO, which is an ethoxylated non-ionic fluorosurfactant, x is an integer from 0 to about 15, and y is an integer from about 1 to about 7. For ZONYL^® FSN, which is a water soluble, ethoxylated non-ionic fluorosurfactant, x is an integer from 0 to about 25, and y is an integer from about 1 to about 9. It can be constituted of 40% fluorosurfactant, 30% 2-propanol and 30% water. Zonyl^® FSN has high surface activity as well as good chemical and thermal stability for use in acid or alkaline solutions. In particular, Zonyl^® FSN-100 is a fluorosurfactant under the Zonyl^® FSN series to denote a water soluble, ethoxylated non-ionic fluorosurfactant which contains no solvent. For ZONYL^® FS-300, which is an ethoxylated non-ionic fluorosurfactant, x is an integer from 0 to about 50, and y is an integer from about 1 to about 9

[0032] In one embodiment of the present invention, ethoxylated non-ionic fluorosurfactants are used. Examples of ethoxylated non-ionic fluorosurfactant include, but are not limited to ZONYL^® FSO, ZONYL^® FSN, ZONYL^® FSN-IOO, and ZONYL^® FS-300, and Maflon® Lineplus PDM 178, PDMl 79, PDM-179D, and PDM-147.

[0033] Polyoxythylene based surfactant which can also be used in the method described herein can be represented by the general formula R₁-R₂-(CH₂CH₂O)_n -H,

[0034] wherein R₁ is an alkyl, alkenyl or alkynyl group having 10 to 22 carbon atoms, R₂ is - O- or -COO-, and n is between 20 and 35. Examples of polyoxythylene based surfactants include, but are not limited to, polyoxyethylene alkylphenols, ethoxylated aliphatic alcohols, polyoxyethylene esters, sorbitan monooleate (20 EO) (Tween 80®; Imperial Chemical (ICI)); sorbitan monostearate (20 EO) (Tween 60^®; ICI); sorbitan monopalmitate (20 EO) (Tween 40^®; ICI); sorbitan monolaurate (20 EO) (Tween 20®; ICI); dinonylphenol ether (7 EO) (Igepal^® DM 430; Rhone-Poulenc (RP)); nonylphenol ether (6 EO) (Igepal^® CO 530; RP); nonylphenol ether (12 EO) (Igepal^® CO 720; RP); dinonylphenol ether (9 EO) (Igepal^® DM 530; RP); nonylphenol ether (9 EO) (Igepal^® CO 630; RP); nonylphenol ether (4 EO) (Igepal^® CO 430; RP); dodecylphenol ether (5.5 EO) (Igepal^® RC 520; RP); dodecylphenol ether (9.5 EO) (Igepal^® RC 620; RP); dodecylphenol ether (11 EO) (Igepal^® RC 630; RP); nonylphenol ether (9.5 EO) (Syn Fac^® 905; Milliken & Company); or octylphenol ether (10 EO) (Triton^® X-IOO; Rohm & Haas).

[0035] Examples of non- ionic hydro genated surfactant include, but are not limited to, hexaethylene glycol dodecyl ether, phospholipid, copolymers of the polyoxyethylenepolyoxypropylene type (e.g., PLURONIC F-68^®) and polyoxyethylene sorbitan esters.

[0036] Typically, concentration of the non-ionic surfactant in the solution is between about 0.01 wt% to about 0.1 wt%, or between about 0.01 wt% to about 0.08 wt%, or between about 0.01 wt% to about 0.06 wt%, or between about 0.01 wt% to about 0.04 wt%, or between about 0.06 wt% to about 0.1 wt% based on the total weight of the solution. The non-ionic surfactants used herein are adsorbed at the gold surface via unspecifϊc binding, thus forming a monolayer of non- ionic surfactant at the gold surface. For example, it is believed that a non- ionic fluorosurfactant binds to noble metal surface via its oxygen atom or hydroxyl (-OH) group. This can have the effect of exposing the fluorocarbon chains on the outer surface of the fluorosurfactant monolayer. The thickness of the monolayer can be dependent on the length of the non- ionic surfactant used. Most commonly, it might be in the range of about 1 nm to about 5 nm. [0037] Generally, any substrate which comprises a noble metal surface can be used. Noble metal includes silver, palladium, gold, platinum, iridium, osmium, rhodium and ruthenium. In one embodiment, silver, gold, platinum, mixtures thereof or alloys thereof can be used. Examples of noble metal alloys include alloys of platinum and iridium, Pd-Pt, Pd-Rh or Pd- Pt-Rh, to name only a few. In one embodiment, the noble metal is gold or an alloy comprising gold.

[0038] The substrate can be manufactured of any material which can be used in the applications referred to herein. For example, the substrate underlying the noble metal surface can be made of a carbon material, a ceramic, glass, such as soda-lime glass, borosilicate glass, acrylic glass, isinglass (Muscovy-glass), aluminium oxynitride, a metal such as titanium, silver, palladium, gold, a metal oxide, a polymer such as polycarbonate and poly(lactic-co- glycolic acid), or mixtures made of different of the aforementioned materials, to name only a few. In one embodiment, the substrate is gold.

[0039] To form a substrate having a noble metal surface, at least a mono layer of noble metal can be formed on a surface of the substrate thus forming a shell of noble metal. However, it is also possible to have a multilayered noble metal shell. The thickness can be about at least 1 nm to about 1 μm or several micrometers. In general, the noble metal layer can be formed using methods known in the art. Examples of such methods include, but are not limited to electroplating, dip coating, spin coating, sputtering, pulsed laser deposition (PLD), physical vapour deposition (PVD) and chemical vapour deposition (CVD).

[0040] The substrate can be in the form of a flat sheet or thin film, a finished article of various shapes or a nano structured material. A nano structured material or nanostructure refers to a structure with dimensions in the nanometer range. Nano structures can be classified into the following dimensional types: Zero dimensional (OD): nanoparticles;

One dimensional (ID): nanorods, nanowires (also called nano fibers) and nanotubes; and Two dimensional (2D): nanoflakes, nano flowers, nanodiscs and nanofilms.

[0041] In one embodiment, the substrate is a nanoparticle. The nanoparticle can be a noble metal nanoparticle or a nanoparticle having a noble metal shell. Typically the size of the nanoparticle is between about 5 nm to 100 nm, or between about 5 nm to 80 nm, or between about 5 nm to 60 nm, or between about 5 nm to 40 nm, or between about 5 nm to 20 nm, or between about 10 nm to 20 nm. Since the dimensions of a nanoparticle is not always regular, i.e. perfectly spherical, the above size refers to the maximal dimension of the nanoparticle in any direction. Smaller sizes for the nanoparticles are generally more stable than larger nanoparticles and have a lower tendency to aggregate. In addition, nanoparticles which are smaller sized have generally larger degrees of curvature which can increase surface density of nucleic acid molecules immobilized on its surface.

[0042] The surface density of the nucleic acid molecules can be uniform or non-uniform. It can be dependent on how the non-ionic surfactants are formed on the noble metal surface, which can be influenced by the concentration of non-ionic surfactant in solution and type of non-ionic surfactant used. In one embodiment, non-ionic fluosurfactants can be formed as a uniform layer when the concentration of non- ionic fluorosurfactant in solution is more than or equal to about 0.01 wt%, which can result in a uniform surface density of nucleic acid molecules. It is believed that the fluorocarbon chain hydrophobic end, such as the fluorocarbon chain hydrophobic end in ZONYL^® FSN, plays an important role in forming an ordered structure of surfactant monolayer on a noble metal surface. [0043] Generally, the pH of the solution at which reaction is carried out can be in any range. It can take place in the range of between about 2 to about 13, or between about 2 to about 7, or between about 7 to about 13. In one embodiment, pH of the solution can be in the range of between about 6 to about 8, or about 7. Typically, the temperature at which reaction is carried out is below 100°C. In one embodiment, the temperature is between about 15° to about 100°C, or between about 15°C to about 75°C, or between about 15°C to about 50°C. In one embodiment, temperature at which reaction is carried out can be in the range of about 20°C to about 35⁰C.

[0044] The general principal of the method of immobilizing nucleic acid described herein may be represented by the general scheme as illustrated for example in Figure l(a). As shown in Figure l(a), a substrate having a noble metal surface, wherein the noble metal surface comprises a layer of non- ionic surfactant, is contacted with a solution comprising nucleic acid molecules. Each nucleic acid molecule can comprise a chemical linker or a chemical linker and a spacer adapted to interact with the noble metal surface. The chemical linker can be bound directly to the nucleic acid or via the spacer. The nucleic acid molecules bind to the noble metal surface after contacting the noble metal surface. In one embodiment, the nucleic acid molecules comprise a chemical linker, and the nucleic acid molecules are immobilized at the noble metal surface via the chemical linker. For example, the chemical linker can be one or two or more thiol groups and the nucleic acid molecules can be immobilized on the noble metal surface via formation of a thiol-noble metal bond with the noble metal surface. In the embodiment illustrated in Fig. 1 (b), the substrate is a nanoparticle. Curvature present on the surface of the nanoparticle may result in greater extent of immobilization of nucleic acid probes on its surface due to lesser extent of steric hindrance by neighboring probes and/or non- ionic surfactant molecules.

[0045] In one embodiment, the method of immobilizing nucleic acid is carried out without sonication. Sonication is the act of applying ultrasound energy to provide agitation to a sample. For example, sonication may be carried out to destabilize nucleic acid molecules which are non-specifϊcally bound on the noble metal surface, which will allow better conformational control of nucleic acid molecules. However, the method described herein can be carried out without sonication. This can be the result of a lower degree of non-specific binding of nucleic molecules on the noble metal surface. Therefore, sonication is not necessary to destabilize the non- specifically bound nucleic acid molecules so as to free up the originally occupied space for specific binding of nucleic acid molecules. [0046] The method of the present invention can be carried out in the presence of salt or without salt. Addition of salt can increase the loading density of nucleic acid on the substrate. Therefore, in one embodiment, the method of immobilizing nucleic acid is carried out in presence of a salt. Salt refers herein to a compound formed by replacing hydrogen in an acid by a metal. The salt can comprise monovalent or divalent cation. The term "monovalent salt" is used herein to mean a water-soluble salt which contains monovalent cations. Examples of a monovalent cation include, but are not limited to, potassium, sodium or lithium. The term "divalent salt" is used herein to mean a water-soluble salt which contains divalent cation. An example of a divalent cation is magnesium. In general, concentration of salt in the solution can be between about 0.05 M to about 1 M, or between about 0.05 M to about 0.8 M, or between about 0.05 M to about 0.6 M, or between about 0.05 M to about 0.4 M, or between about 0.8 M to about 1.0 M, or between about 0.6 M to about 0.8 M. Concentration of salt in the solution can affect the loading density of nucleic acid on the substrate. Therefore, reaction conditions for the above method are adapted such as to form substrates with a specific loading density of nucleic acid with good conformational control. In one embodiment, a non-ionic hydro genated surfactant, such as hyexaethylene glycol dodecyl ether, is used whereby the concentration of salt in solution is between about 0.01 M to about 0.2 M.

[0047] In a second aspect the invention provides a method of detecting a target nucleic acid. The method includes immobilizing a capture nucleic acid adapted to hybridize to the target nucleic acid on a noble metal surface of a substrate, wherein a no n- ionic surfactant is immobilized at the noble metal surface. In a further step, the method includes contacting a sample suspected to contain the target nucleic acid with the substrate.

[0048] In another aspect the invention refers to a substrate obtained by a method referred to herein or a kit comprising a substrate obtained by a method referred to herein. The substrate comprising nucleic acid can be used, for example, as a biosensor, DNA biosensor microarray, novel molecular devices and gene chip.

[0049] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0050] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0051] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0052] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples. It is understood that modification of detail may be made without departing from the scope of the invention. EXEMPLARY EMBODIMENTS OF THE INVENTION

Materials

[0053] Hydrogen tetrachloroaurate(III) trihydrate, with formula HAuCl₄" 3H₂O, trisodium citrate, silver nitrate, Zonyl® FSN-100, with formula F(CF₂CF₂)_3-8CH₂CH₂O(CH₂CH₂O)_0-i₅H (FSN), tris(2-carboxyethyl)phosphine (TCEP), and dithiothreitol (DTT) were purchased from Sigma-Aldrich. All other reagents of certified analytical grade were used as received. Oligonucleotides were obtained from 1^st Base Pte Ltd (Singapore), as listed in Table 1. Colloid solutions of 100 nm gold nanoparticles were obtained from Ted Pella, Inc. (Redding CA). OliGreen ssDNA Quantification Kit was purchased from Invitrogen (Singapore).

Table 1

Characterization

[0054] Quantification of ssDNA samples were performed with a NanoDrop TM 1000 Spectrophotometer (Thermo Scientific). Absorption spectra of gold nanoparticles colloids were collected using an Agilent Gl 103 A UV-Vis Spectrophotometer. Fluorescence measurements were performed on a microplate spectrofluorometer (Safire2 Microplate Reader, Tecan Group Ltd, Switzerland). An Eppendorf centrifuge 5415R was used for centrifugation of the gold nanoparticles. High-resolution transmission electron microscopy (HR-TEM) images of the gold nanoparticles and the ssDNA-gold nanoparticle conjugates were collected using an FEI Tecnai TF-20 field emission HR-TEM at 200 kV.

Example 1: Preparation of gold nanoparticles

[0055] Gold nanoparticles can be prepared using various methods reported. One method is using reduction of chloroauric acid (HAuCl₄) with sodium citrate.

[0056] In a typical process, all glassware used for the preparation of gold nanoparticles was washed with freshly prepared aqua regia, which is a mixture made up of nitric acid and hydrochloric acid in a volumetric ratio of 1:3 (i.e. HNO₃:HC1=1 :3). Subsequently, the glassware is rinsed with ultrahigh purity water and dried in an oven at 100 °C for 2 to 3 hours.

[0057] 60 ml solutions of 0.01 % chloroauric acid were made to boil under stirring in a round-bottom flask fitted with a reflux condenser. Subsequently, 4.5ml and 0.6ml of 1.0 wt% sodium citrate were each added to chloroauric acid to obtain colloid solutions of gold nanoparticles with average diameters of 13nm and 40nm respectively. The reaction mixtures were maintained at boiling point under continuous stirring for about 15 minutes, afterwhich they were cooled to room temperature and stored at 4°C prior to use.

[0058] With the assumption that the gold nanoparticles are spherical and have a density value equivalent to bulk gold of 19.30 g/cm³, concentration and extinction coefficients (E) of surface plasmon resonance (SPR) absorption of the gold nanoparticles in solution were calculated. Concentration of 13-nm gold nanoparticles solution was calculated to be 4.03 nM with ε = 2.08 x 10⁸ L/(mol^#cm) at 524 nm. Concentration for 40-nm gold nanoparticles were calculated to be 0.14 nM with E = 8.57 x 10⁹ L/(mol^«cm) at 530 nm.

[0059] For the 100-nm gold nanoparticles purchased from Ted Pella, concentration information was obtained from the supplier at 0.008 nM. E was calculated to be 1.02 x 10¹¹ L/(mol^»cm) at 573 nm.

[0060] Capping of gold nanoparticles with fluorosurfactant Zonyl® FSN-100 was carried out by adding about 0.05 wt% Zonyl® FSN-100 to the colloid solutions of gold nanoparticles to form FSN-stabilized gold nanoparticles (FSN used herein to denote FSN-100).

Example 2: Preparation of ssDNA-modified FSN-stabilized gold nanoparticles [0061] Typically, samples of thiolated ssDNA (i.e. Probe 1, Probe 1*, Probe 2, Probe 3 and Probe 4) were mixed with separate portions of 0.1 M dithiothreitol in 0.2 M phosphate buffered saline (PBS) having a pH of 8.0 for 1 hour, and purified using an NAP-5 column (GE Healthcare). The purified thiolated ssDNA samples were stored at 4 °C prior to use. To avoid disulfide formation between the thiolated ssDNA strands, the purified thiolated ssDNA samples were incubated with 5 mM of tris(2-carboxyethyl)phosphine, which has a pH of 7.5, for 10 minutes before mixing with the FSN-stabilized gold nanoparticles.

[0062] Unless stated otherwise, the mixture solutions, which contain about 2 μM of thiolated ssDNA, about 2 nM of FSN-stabilized gold nanoparticles, 10 mM phosphate buffer of pH 7.5 and sodium chloride (NaCl) of stipulated concentration of up to 1.0 M, were incubated at room temperature for 2 hours.

[0063] Following incubation, excess ssDNA was removed by centrifugation at 13.2 K rpm for 15 minutes. Samples of the ssDN A-nanoparticle conjugates formed were re-suspended in 0.1 M phosphate buffered saline of pH 7.5 to form conjugate solutions of 2nM. This process was repeated at least five times for each sample to wash off unreacted ssDN A. In addition, fluorescence measurement was carried out as a check to ensure that there was no free ssDNA in the supernatant following the last washing cycle.

[0064] Generally, non-thiolated ssDNA (Le. Probe 1 '*) modified FSN-stabilized gold nanoparticles can also be formed in the same manner as described above.

Example 3: Quantification of oligonucleotides loaded on gold nanoparticles

[0065] Concentration of the nanoparticle conjugate solutions can be calculated using Beer's Law, i.e.

A = Slc

where A = absorbance of the sample, S = molar absorbtivity (L/(mol^»cm)), / = path length (cm), and c = concentration of the nanoparticles in solution (M). Absorbance of the solution was measured using UV-visible spectrophotometer.

[0066] To determine the amount of thiolated ssDNA loaded on gold nanoparticles, dithiothreitol displacement was carried out. Equal volumes of the thiolated ssDNA-gold nanoparticle conjugates and 1.0 M dithiothreitol in 0.2 M phosphate buffer of pH 8.0 were mixed and incubated for 16 hours. This caused the surface-bound ssDNA molecules to be chemically displaced from the surface of the nanoparticles. Aggregation of the gold nanoparticles results. The gold aggregates were removed by centrifugation at 10.0 K rpm for lO min.

[0067] For the 6'FAM-labeled thiolated ssDNA samples (i.e. Probe 1*), 100 μL of the ssDNA supernatant was placed in a 96-well plate and fluorescence measurements were carried out. For the unlabeled thiolated ssDNA samples (i.e. Probe 1, Probe 2, Probe 3 and

Probe 4), OliGreen ssDNA Quantification Kit was used. A standard calibration curve was obtained at the same time using ssDNA samples prepared with the same dithiothreitol buffer solution. The results obtained using the two quantification methods are in agreement with each other. AU measurements were performed at least three times to ensure repeatability.

Example 4: Hybridization of ssDNA targets to ssDNA probe-gold nanoparticle conjugates

[0068] To examine the hybridization behavior of thiolated ssDNA-functionalized gold nanoparticles, each of the thiolated ssDNA-nanoparticle conjugate solution of 2 nM was split into 2 portions. Conjugated solutions formed from unlabeled ssDNA probes, i.e. Probe 1, Probe 2, Probe 3 and Probe 4, were used. One portion of each solution was used to determine the loading density of the probes following the dithiothreitol displacement. The other portion was incubated at room temperature for 3 hours with 1 μM target oligonucleotides (i.e. Target, which is labelled with 6'FAM) that were complementary to the nanoparticle-bound probes, under a hybridization condition of 0.2 M sodium chloride and 10 mM phosphate with pH 7.5. After hybridization, the free non-hybridized targets were removed by centrifugation and rinsing with 0.1 M PBS with pH 7.5 for at least five times. Fluorescence measurements were also carried out as a check to ensure that there was no free non-hybridized target in the supernatant following the last washing cycle.

[0069] The non-specifically adsorbed ssDNA, ssDNA probes and hybridized DNA duplexes, were displaced by dithiothreitol. Since only the targets were 6'FAM-labeled, concentration of the DNA duplexes were determined by fluorescence measurements.

Example 5: Effect of sodium chloride on thiolated ssDNA loading on gold nanoparticles

[0070] Figure 2(a) - (d) shows the loading profiles of various thiolated ssDNA probes on 13- ran gold nanoparticles as a function of sodium chloride concentration in the immobilization solution. Figure 2(a) shows the loading profile of thiolated ssDNA bearing a TlO spacer (Probe 1). Figure 2(b) shows the loading profile of thiolated ssDNA bearing a T20 spacer (Probe 2). Figure 2(c) shows the loading profile of thiolated ssDNA bearing a iSplδ spacer (Probe 3). Figure 2(d) shows the loading profile of thiolated ssDNA with iSplδ and T20 spacer (Probe 4).

[0071] All figures show that an increase of sodium chloride concentration led to a higher loading density of the ssDNA. This may mainly be attributed to the screening effect of the concentrated counter ions on the electrostatic repulsion force between the surface-bound ssDNA strands. Table 2 shows a comparison table of surface coverage of thiolated ssDNA on gold nanoparticles.

Table 2

Gold Nanoparticle Probe Spacer Condition ssDNA Surface

Size (nm) No. Strands/ coverage nanoparticle (pmol/cm²)

13 1 T₁₀ 1. OM NaCl 110 34

13 1 T₁₀ LOM NaCl + 132 41

5OnM Mg²⁺

13 3 iSpl8 1. OM NaCl 170 53

13 2 T₂₀ 1. OM NaCl 95 30

13 4 iSpl8+T₂₀ 1. OM NaCl 102 32

40 1 Ti₀ LOM NaCl 570 19

100 1 Ti₀ LOM NaCl 2807 15

[0072] In the presence of 1.0 M sodium chloride, FSN-stabilised gold nanoparticle conjugates with about 110 strands of Probe 1 per 13 nm particle. This corresponds to a surface coverage of about 34 pmol/cm². Likewise, in the presence of 1.0 M sodium chloride, FSN- stabilised gold nanoparticle conjugates with about 95 strands of Probe 2 per 13 nm particle, 170 strands of Probe 3 per 13 nm particle and 102 strands of Probe 4 per 13 nm particle. The surface coverage for Probe 2, 3 and 4 in the presence of 1.0 M sodium chloride are about 30, 53 and 32 pmol/cm² respectively.

[0073] The results show that, through an embodiment of the present invention, loading of ssDNA on gold nanoparticles can be achieved in one single step, simply by mixing thiolated ssDNA with solution containing FSN-stabilized gold nanoparticles and allowing the mixture to incubate for a few hours, even in the presence of sodium chloride with concentration of up to 1.0M and over a wide pH range from 2 to 13. This is an improvement over state of the art methods for the preparation of ssDNA-gold nanoparticle conjugates, one of which includes use of a slow salt-aging process that involves several steps and may take a day to prepare. The slow salt-aging process is required because commonly used citrate-stabilized gold nanoparticles are only dispersed stably in aqueous solutions with low ionic strength, and presence of more than 50 mM of sodium chloride in the solution may induce irreversible aggregation of the nanoparticles.

Example 6: Effect of sonification on thiolated ssDNA loading on gold nanoparticles

[0074] It was previously reported in literature that sonication of the immobilization solution would result in a higher ssDNA surface coverage, which is attributed to destabilization of the ssDNA nonspecific binding, thus allowing more thiolated ssDNA to be attached via the sulfur-gold interaction. However, no obvious effect of sonication was observed in the present application. In the presence of 1.0 M sodium chloride, FSN-stabilized gold nanoparticle conjugates with about 106 strands of Probe 1 per 13 nm nanoparticle with sonication, and with about 110 strands of Probe 1 per 13 nm nanoparticle without sonication. This can be due to low non-specific adsorption of thiolated ssDNA on the FSN-modified gold nanoparticle surface.

Example 7: Effect of FSN-stabilized gold nanoparticles on ssDNA adsorption

[0075] Experiments were carried out to investigate effects of fluorosurfactants on adsorption of ssDNA on gold nanoparticles.

[0076] Probe 1'* (6 'FAM- labeled, non-thiolated) was used in the experiments to investigate non-specific adsorption behaviour of ssDNA on citrate-stablized and FSN-stablized 13 nm gold nanoparticles. Figure 3 shows a comparison graph of surface density of non-specifically adsorbed ssDNA (non-thiolated) the gold nanoparticles. The graph shows a much lower level of non-specifically adsorbed ssDNA (non-thiolated) on FSN-stabilized gold nanoparticles compared to that of citrate-stablilized gold nanoparticles. In addition, the inventors have found this inhibiting effect of fluorosurfactant against adsorption of ssDNA is independent of the ionic strength of the solution.

[0077] Interaction between ssDNA and gold surfaces may occur because, although ssDNA is coiled in its native configuration when the negatively charged backbone is exposed to the aqueous solution, transient structural fluctuations may uncoil the ssDNA and allow attachment of nucleobases to the negatively charged gold nanoparticles. It has also been reported by Kimura-Suda et al in J.Am Chem. Soc. 2003, 125, 9014-9015 that the attachment of nucleobases on gold surfaces may be strong enough to compete with thiol-gold linkage. However, through an embodiment of the present invention, it has been demonstrated that undesirable non-specific ssDNA loading can be suppressed effectively when gold nanoparticles are stabilized by fluorosurfactant, in which the fluorosurfactant adlayer serves to prevent nucleobases of ssDNA from interacting with the gold nanoparticle surface

[0078] Probe 1 and 1* were used in the experiments to investigate loading profile of thiolated ssDNA on 13-nm gold nanoparticles, and which was carried out using ex situ quantification of the surface-bound ssDNA as a function of the incubation time following dithiothreitol displacement. Figure 4(a) is a chart showing the surface density of thiolated ssDNA (Probe 1) on 13-nm gold nanoparticle as a function of incubation time. It can be seen that a saturated ssDNA layer was formed on the gold nanoparticle within two hours. In situ monitoring by fluorescence measurement was also carried out using Probe 1* (6'FAM-labeled). Figure 4(b) is a chart showing fluorescence evolution of the mixture solution of the ssDNA (Probe 1*) and the FSN-stabilized gold nanoparticles as a function of incubation time. It can be seen that fluorescence intensity of the solution decreased gradually after mixing, as a result of quenching by gold nanoparticles upon ssDNA immobilization. No further decrease of fluorescence intensity of the solution was observed after about 120 minutes (i.e. two hours), which signifies a saturated ssDNA functional layer formation.

[0079] For comparison, the FAM-labeled non-thiolated ssDNA (i.e. Probe 1'*) was also mixed with FSN-stabilized gold nanoparticles, and fluorescence emission was monitored. In this case, the fluorescence signal showed no obvious change over several hours. Figure 5 is a graph showing fluorescence evolution of the mixture solution of the FAM-labeled non- thiolated ssDNA (Probe 1 '*) and the 13 nm FSN-stabilized gold nanoparticles as a function of incubation time. It can be seen from the graph that fluorescence intensity of the solution did not show a change after mixing. This signifies that non-thiolated ssDNA cannot be immobilized through non-specific adsorption.

[0080] As demonstrated, both ex situ and in situ measurements of the loading kinetics of thiolated ssDNA on FSN-stabilized gold nanoparticles indicate that the fluorosurfactant adlayer allows rapid attachment of ssDNA through the thiol-gold linkage. Because the interaction between the nucleobases and the gold surface was inhibited, no slow reorganization of molecular configuration occurred in the adsorption process. The thiolated ssDNA molecules simply displaced the adsorbed fluorosurfactant species and were immobilized at an upright orientation. Therefore, by using an embodiment of the present invention, control of ssDNA conformation could be realized during the immobilization process. This removes the need for sonication or further chemical treatment with mercaptohexanol which are required in conventional methods, which significantly reduces the time required for formation of the ssDNA-gold nanoparticle conjugates.

Example 8: Effect of divalent cations on ssDNA adsorption

[0081] Maximum loading density of thiolated ssDNA (Probe 1) was increased by more than 20% from a surface coverage of 34 to 41 pmol/cm² when 50 mM of magnesium divalent cations (Mg²⁺) was present in the immobilization solution containing 1.0 M sodium chloride. This indicates that the ability of divalent cations of Mg²⁺ to more efficiently screen the electrostatic repulsion between surface-bound ssDNA molecules. This is surprising because it has been reported previously by other groups, for example Hurst et al (Anal. Chem. 2006, 78, 8313-8318), that divalent cations may induce rapidly irreversible aggregation of gold nanoparticles. In an embodiment of the present invention, FSN-stabilized gold nanoparticles were stable enough to withstand presence Of Mg²⁺ with a concentration of up to 0.1 M. The inventors have also repeated the experiments for that of calcium (Ca²⁺) using 10 mM calcium chloride (CaCl₂) and aluminium (Al³⁺) using 10 mM aluminium nitrate (A1(NO₃)₃) and found that they do not work as well, and would still lead to irreversible color change of the FSN- stabilized gold nanoparticle solution from red to blue in about one minute.

Example 9: Effect of Spacer on ssDNA adsorption

[0082] Referring to Table 2, it can be seen that there is a significant effect of spacer (the region between the recognition sequence and the thiol modification site) on the loading density. Maximum coverage of the 12-base ssDNA with an iSplδ spacer (Probe 3) is about 170 strands per 13 run particle, which is more than 1.5 times higher than that of ssDNA with a TlO spacer (Probe 1), which has about 110 strands per 13 nm particle.

[0083] This should be attributed to a more intense packing of the uncharged iSplδ spacer as compared with TlO. However, the loading density of a 32-base ssDNA with an iSplδ spacer

(Probe 4) is much lower, at about 102 strands per 13 nm particle. Various research groups have reported that loading density of thiolated ssDNA becomes lower as the strand length increases, which could be due to increased repulsive interaction of the more flexible long ssDNA strands. The inventors have found that the iSplδ spacer may be able to increase the coverage of the thiolated ssDNA by up to 30 bases.

Example 10: Effect of nanoparticle size on ssDNA adsorption

[0084] Effect of nanoparticle size on ssDNA adsorption was investigated using larger gold nanoparticles with average diameters of about 40nm and about lOOnm for immobilization of ssDNA. It was found that the larger sized gold nanoparticles exhibit excellent stability in aqueous solution after capping with fluorosurfactants, even in the presence of 1.0 M sodium chloride. This is surprising as studies using citrate-stabilized gold nanoparticles have shown that larger sized particles are usually less stable than the smaller ones. Therefore, according to one embodiment of the present invention, use of fluorosurfactants on gold nanoparticles has increased stability of thiolated ssDNA with larger gold nanoparticles. A lower surface density of the thiolated ssDNA at the larger nanoparticles was observed, which could be ascribed to the effect of the surface curvature. Degree of curvature of nanoparticles plays an important role in loading of thiolated ssDNA. There is an increase in surface density of attached ssDNA as diameter of nanoparticle decreases from 60 to 10 nm.

Example 11: Hybridization efficiency of thiolated ssDNA

[0085] Hybridization ability of ssDNA-nanoparticle conjugates with DNA strands that are complementary in sequence to the nanoparticle-attached ssDNA is essential for the function of the conjugates. Figure 6 is a graph showing the hybridization efficiency as a function of surface density of thiolated ssDNA on 13nm gold nanoparticles for thiolated ssDNA bearing TlO (Probe 1), T20 (Probe 2), and iSpl8 (Probe 3) spacers.

[0086] The graph shows that the hybridization efficiency is a function of the surface density of the probes. For the conjugates of Probe 1, an efficiency of more than 60% could be achieved when less than 50 probe strands were loaded on one gold nanoparticle (corresponding to about 15 pmol/cm²). The high hybridization efficiency suggests that most of the thiolated ssDNA molecules may be bound with an upright orientation and are available to hybridize with their targets, since hybridization efficiency generally drops with an increase in surface density of thiolated ssDNA on nanoparticle. This is because high surface density of the probes leads to an increase in repulsive interaction (both electrostatic and steric) between the probe ssDNA and its complementary target.

[0087] From Figure 6, hybridization behavior was also found to be sensitive to spacer length of the thiolated ssDNA. At similar surface densities, hybridization efficiency decreased as the spacer of the thiolated ssDNA became shorter. As an example, when the probe loading density was 100 strands per nanoparticle, the conjugates of Probe 2, which has a spacer of T20, exhibited the highest hybridization efficiency of about 40%. However, this value decreases significantly to about 1% for the conjugates of Probe 3, which has an iSpl8 spacer. These results indicate that, although use of the iSplδ spacer has demonstrated a high ssDNA loading of about 170 strands per 13 nm nanoparticle, it was difficult for the target ssDNA to hybridize with the surface-bound ssDNA, in particular when the surface coverage is larger than 100 strands per 13 nm nanoparticle. Therefore, there exists a trade-off between probe loading density and hybridization efficiency.

[0088] As a control, the conjugates were mixed with the non-complementary ssDNA, and no detectable amount of the ssDNA was attached to the conjugates under the hybridization condition. In addition, for comparison, the conjugates of Probe 3, which has an iSpl8 spacer, was also prepared following the slow salt-aging process known in literature at a final sodium chloride concentration of 1.0 M with no fluorosurfactant used. Table 3 summarizes the hybridization behavior of Probe 3 on 13nm gold nanoparticle with no fluorosurfactant. Results indicate that lower hybridization efficiency of the conjugates of the shorter probes was not influenced by fluorosurfactant on the surfaces of the nanoparticles.

Table 3

[0089] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0090] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0091] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognised that various modifications are possible within the scope of the invention claimed. Additional objects, advantages, and features of this invention will become apparent to those skilled in the art upon examination of the foregoing examples and the appended claims. Thus, it should be understood that although the present invention is specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

ClaimsWhat is claimed is:

1. A method of immobilizing a nucleic acid on a substrate having a noble metal surface, the method comprising contacting the nucleic acid with the substrate, wherein the substrate comprises a non-ionic surfactant on its noble metal surface.

2. The method of claim 1, wherein the non- ionic surfactant is selected from the group consisting of a fluorosurfactant, a polyoxyethylene based surfactant, octyl glucoside, and a sorbitan ester.

3. The method of claim 2, wherein the fluorosurfactant is an ethoxylated non- ionic fluorosurfactant.

4. The method of claim 3, wherein the ethoxylated non-ionic fluorosurfactant is of the general formula:

Rf-CH₂CH₂O(CH₂CH₂O)_xH

wherein Rf has the general formula F(CF₂CF₂)_y, and wherein x is an integer from about 0 to about 50, and y is an integer from about 1 to about 9.

5. The method of any of claims 1 to 4, wherein the substrate is a nanoparticle.

6. The method of claim 5, wherein the nanoparticle is a noble metal nanoparticle or a nanoparticle having a noble metal shell.

7. The method of any of claims 1 to 6, wherein the non- ionic surfactant is contacted with the substrate having a noble metal surface after synthesis of the noble metal surface.

8. The method of any one of the preceding claims, wherein the noble metal is gold, or silver or platinum.

9. The method of any one of the preceding claims, wherein the nucleic acid comprises a chemical linker adapted to interact with the noble metal surface.

10. The method of claim 9, wherein the chemical linker is a thiol group.

11. The method of any one of the preceding claims, wherein the nucleic acid comprises a spacer.

12. The method of claim 11, wherein the spacer is an adenosine spacer, or a thymine spacer or a polyethylene glycol (PEG) spacer.

13. The method of any one of the preceding claims, wherein the nucleic acid is selected from the group of a DNA, PNA or RNA molecule.

14. The method of any one of the preceding claims, wherein the method is carried out without sonication.

15. The method of any one of the preceding claims, wherein contacting the nucleic acid with the substrate is carried out in presence of a salt.

16. The method of claim 15, wherein the salt comprises a monovalent or divalent cation.

17. The method of claim 16, wherein the monovalent cation is potassium or sodium or lithium.

18. The method of claim 16, wherein the divalent cation is magnesium.

19. The method of any one of the preceding claims, wherein contacting the nucleic acid with the substrate is carried out in an aqueous solution, wherein the amount of the non-ionic surfactant in the solution is between about 0.01 to about 0.1 wt% based on the total weight of the solution.

20. The method of claim 15 to 18, wherein contacting the nucleic acid with the substrate is carried out in an aqueous solution, wherein the concentration of the salt in the solution is between about 0.05 M to about 1 M.

21. The method of claim 20, wherein the concentration of the salt in the solution is between about 0.01 M to about 0.2 M in case the non-ionic surfactant is a hydrogenated surfactant.

22. The method of claim 21, wherein the hydrogenated surfactant is selected from the group consisting of hexaethylene glycol dodecyl ether, phospholipid, copolymers of the polyoxyethylenepolyoxypropylene type and polyoxyethylene sorbitan esters.

23. The method of claim 5 or 6, wherein the maximum dimension of the nanoparticle is between about 5 to 100 nm.

24. A method of detecting a target nucleic acid, the method comprising:

- immobilizing a capture nucleic acid adapted to hybridize to the target nucleic acid on a noble metal surface of a substrate, wherein a non-ionic surfactant is immobilized on the noble metal surface; and ■ contacting a sample suspected to contain the target nucleic acid with the substrate.

25. The method of claim 24, wherein the capture nucleic acid comprises a nucleic acid sequence that is at least in part substantially complimentary to a part of the nucleic acid sequence of the target nucleic acid.

26. A substrate for detecting a target nucleic acid, the substrate comprising a noble metal surface, wherein the substrate has immobilized on said noble metal surface a non-ionic surfactant and a capture molecule adapted to hybridize to the target nucleic acid.

27. The substrate of claim 26, wherein the substrate is a nanoparticle.

28. A kit for detecting a target nucleic acid, the kit comprising a substrate comprising a noble metal surface, wherein the substrate has immobilized on said noble metal surface a non-ionic surfactant and a capture molecule, wherein the capture molecule is adapted to hybridize to the target nucleic acid.