WO2013185167A1

WO2013185167A1 - Metallic nanoparticle treated cellulosic substrate as a sers biodiagnostic platform

Info

Publication number: WO2013185167A1
Application number: PCT/AU2013/000574
Authority: WO
Inventors: Gil Garnier; Ying Hui NGO; Whui Lyn THEN; George Simon; Dan Li
Original assignee: Monash University
Priority date: 2012-06-13
Filing date: 2013-05-31
Publication date: 2013-12-19

Abstract

A metal nanoparticle treated cellulosic substrate for use as a surface enhanced Raman scattering (SERS) biodiagnostic platform, the substrate including metal nanoparticles coating the substrate, wherein the nanoparticles are agglomerated on the substrate due to pre-treatment with a coagulant.

Description

METALLIC NANOPARTICLE TREATED CELLULOSIC SUBSTRATE AS A SERS BIODIAGNOSTIC PLATFORM

FIELD OF THE INVENTION

[0001 ] The invention is generally directed to Surface Enhanced Raman Scattering (SERS) substrates suitable for use as a SERS biodiagnostic platform, and in particular to metallic nanoparticle treated substrates formed of paper or other cellulosic material.

BACKGROUND TO THE INVENTION

[0002] Paper has recently been rediscovered as substrate for low cost analytical tests in health and environmental applications. Flexible micro-fluidic systems, reactors and valves have already been printed on paper to regulate, measure and control the flow of analytes and reagents. In paper blood typing assays, blood group antigens can be displayed directly by the red blood cells that elude or absorb on the paper depending on whether they react with a specific or a non-specific antibody which was previously adsorbed. However, the identification of antibodies can be complicated because of their typical low concentration and small dimensions, such as for example immunoglobulin G (IgG). While Enzyme- Linked Immunosorbent Assay (ELISA) is a standard solution-based technique, its application to paper, while possible, requires multiple reactants and washing steps. A direct approach involving a simple contact between a paper substrate and analytes, followed by signal amplification using some instrumentation techniques would be ideal. Surface Enhanced Raman Scattering (SERS) presents an attractive technique where analytes such as antibodies can be selectively adsorbed onto antigens capped metallic nanoparticles on a treated paper substrate and detected at very low concentrations. [0003] SERS is a technique that enhances the Raman scattering which measures the small energy changes of the light scattered from a molecule absorbed on a metallic surface, typically a metallic nanoparticle. The

enhancement factor can be as much as 10¹⁴-10¹⁵, which enables SERS as a single-molecule detection technique to identify analytes at trace levels. The critical requirements of a substrate for SERS application in routine analytical procedures include low cost, sensitivity, robustness, reproducibility and stability to allow for long term storage between measurements. The immobilization of metallic nanoparticles onto solid substrates provides an attractive alternative to metal aqueous colloid as SERS substrates because of its higher flexibility and easy application to non water-soluble compounds. In particular, there has been significant progress in surface sciences and nanofabrication technology that have allowed the control of size, shape and aggregation of nanoparticles deposited on conventional SERS substrates such as silicon and glass by electron beam lithography, focused ion beam patterning and thermal evaporation. However, these techniques are time consuming, costly, require sophisticated equipment, and the substrates are often fragile and suffer from poor storage stability.

[0004] With excellent features such as low cost, flexibility, robustness, long term stability and ease of use, cellulosic materials such as paper offers a promising platform as a SERS substrate for bioassays. The soft texture of paper substrates allows conformal contact with the surface of analytes by swabbing. This provides an efficient and practical method to collect traces of analytes from the body and many surfaces, compared to silicon and glass which are rigid, brittle and confined to laboratory experiments. Paper is an efficient substrate for routine SERS analysis as highlighted in many studies. However, most of these studies have used paper simply as an inert support and have not explored how the rough, composite and heterogeneous nature of paper can affect the distribution of nanoparticles and the SERS signal generated.

[0005] Paper was first reported as a SERS substrate in 1984 by Tran [Tran, CD., Subnanogram detection of dyes on filter paper by surface-enhanced Raman scattering spectrometry. Analytical Chemistry, 1984. 56(4): p. 824-826] which demonstrated subnanogram detection of dyes on filter paper. Silver colloidal hydrosols were synthesized. Various concentrations of aqueous dye solutions and silver colloidal were applied onto filter paper by microsyringe, either as premixed mixture or separately. The Raman spectra were taken on wet paper.

[0006] Vo-Dinh [Vo-Dinh, T., M. Uziel, and A.L. Morrison, Surface-Enhanced Raman Analysis of Benzo[A]Pyrene-DNA Adducts on Silver-Coated Cellulose Substrates. Appl. Spectrosc, 1987. 41 (4): p. 605-610] and Laserna [Berthod, A., J.J. Laserna, and J.D. Winefordner, Analysis by surface enhanced Raman spectroscopy on silver hydrosols and silver coated filter papers. Journal of Pharmaceutical and Biomedical Analysis, 1988. 6(6-8): p. 599-608] [Cabalin, L.M. and J.J. Laserna, Fast spatially resolved surface-enhanced Raman spectrometry on a silver coated filter paper using charge-coupled device detection. Analytica Chimica Acta, 1995. 310(2): p. 337-345] [Bizzarri, M.S.P.A.R. and M.S.P.S.

Cannistraro, SERS detection of thrombin by protein recognition using

functionalized gold nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 2007. 3(4): p. 306-310] reported the assembly, morphology and optical properties of AgNPs-coated filter paper as a promising SERS substrate. The AgNPs coated papers were prepared by immersing the filter paper in AgN03 solution and the wet filter paper was sprayed with sodium borohydride solution by thermal evaporation in a vacuum chamber. These filter-paper had a random dispersion of particle sizes and shapes.

[0007] Luo [Luo, Z. and Y. Fang, SERS of C60/C70 on gold-coated filter paper or filter film influenced by the gold thickness. Journal of Colloid and Interface Science, 2005. 283(2): p. 459-463], Niu [Niu, Z. and Y. Fang, Surface-enhanced Raman scattering of single-walled carbon nanotubes on silver-coated and gold- coated filter paper. Journal of Colloid and Interface Science, 2006. 303(1 ): p. 224- 228], Wu. [Wu, D. and Y. Fang, The adsorption behavior of p-hydroxybenzoic acid on a silver-coated filter paper by surface enhanced Raman scattering.

Journal of Colloid and Interface Science, 2003. 265(2): p. 234-238] and Ma. [Ma, W. and Y. Fang, Experimental (SERS) and theoretical (DFT) studies on the adsorption of p- m-, and o-nitroaniline on gold nanoparticles. Journal of Colloid and Interface Science, 2006. 303(1 ): p. 1 -8.] performed direct synthesis of gold and silver colloids by chemical oxidation-reduction using potassium

tetrachloroaurate (KAuCI₄) and silver nitrate (AgN0₃). The resulting colloids were added drop wise onto filter papers for SERS application. Nanoparticles coated paper was shown a highly SERS active substrate. However, the spotting method used did not achieve reproducible and uniform distribution of nanoparticles on paper; aggregation is formed at the edge of the droplets during drying (coffee stain phenomenon).

[0008] International Publication No. WO 2010/073260 describes a SERS active paper substrate comprising in-situ synthesized nanoparticles. The nanoparticles paper was prepared by immersing the paper strip in a metal precursor solution and then immersion in a boiling reducing solution. However, the process used is not practical.

[0009] Yu and White [Yu, W.W. and I.M. White, Inkjet Printed Surface

Enhanced Raman Spectroscopy Array on Cellulose Paper. Analytical Chemistry, 2010. 82(23): p. 9626-9630] printed silver nanoparticle on hydrophobic paper with an inkjet printer. This method to fabricate SERS paper involved tedious pre- concentration of the silver ink and required multiple printing to produce adequate amount of nanoparticles on paper. This is not practical.

[0010] Cheng [Cheng, .-L, B.-C. Tsai, and J. Yang, Silver nanoparticle- treated filter paper as a highly sensitive surface-enhanced Raman scattering (SERS) substrate for detection of tyrosine in aqueous solution. Analytica Chimica Acta, 201 1 . 708(1-2): p. 89-96] prepared silver nanoparticle doped filter papers with a silver mirror reaction. Gold nanorod loaded filter paper was also demonstrated by Lee. [Lee, C.H., L. Tian, and S. Singamaneni, Paper-Based SERS Swab for Rapid Trace Detection on Real-World Surfaces. ACS Applied Materials & Interfaces, 2010. 2(12): p. 3429-3435; Lee, C.H., et al., Highly Sensitive Surface Enhanced Raman Scattering Substrates Based on Filter Paper Loaded with Plasmonic Nanostructures. Analytical Chemistry, 201 1 . 83(23): p. 8953-8958] by dipping paper in a gold nanorod suspension synthesized by a seed-mediated approach.

[001 1 ] US Patent Publication No. 2010/0245814 describes an analytical substrate where a film having metal nanoparticles is deposited on a substrate to form a metallic film thereon for amplifying Raman signals.

SUMMARY OF THE INVENTION

[0012] It would be advantageous to have a metal nanoparticle treated cellulosic substrate which provides improved SERS performance over known SERS substrates.

[0013] It would also be preferable that the SERS substrate not lose efficiency following wetting during usage.

[0014] According to one aspect of the present invention there is provided a metal nanoparticle treated cellulosic substrate for use as a surface enhanced Raman scattering (SERS) biodiagnostic platform, the substrate including metal nanoparticles coating the substrate, wherein the nanoparticles are agglomerated on the substrate due to pre-treatment with a coagulant.

[0015] This invention may be used as a solid, flexible and conformable substrate platform for SERS analysis for health, analytical and environment application. The applicants have determined that the aggregation state of the nanoparticles is the main contributing factor to SERS performance. Furthermore, the nanoparticles can be controlled by use of a coagulant to control the distribution of the nanoparticles on the substrate surface.

[0016] The substrate may be formed from paper. The term 'paper' refers to a non-woven composite made from cellulosic fibers. These fibers may include pulp fibers, from mechanical or chemical pulping process, from hardwood or hardwood sources, wood or non-wood sources (hemp, bagasse, flax), cotton fibers, nanocellulose and micro fibers. The paper non-woven composite may have a basis weight ranging from 15 to 500 g/m2, and better 40- 400 g/m2. Paper is a non-woven material either made from wet-process or by air-laid process.

[0017] The coagulant used to agglomerate the nanoparticles may preferably be a polymer. These polymers may include high molecular weight (100,000 Dalton to 20,000,000 Dalton), high charge density (5% to 1 00% based on monomer charged) polymers. The polymer can have a linear, branched, dendritic, alternating or block morphology. The polymers that may be utilised include CPAM, PEI, PEO, PVAm, PAE, polyDADMAC and similar polymers and their copolymers and blends. The polymer can be added to paper either as bulk treatment (during formation of paper by wet end addition, or by dipping paper in a polymer solution) or as a surface treatment (by printing, surface sizing, surface coating, dripping, or spraying). The polymer can also be added to the

nanoparticle suspension to preform aggregates to be subsequently adsorbed on paper. The polymer also acts to retain the nanoparticles on the substrate following wetting during usage such that there is little to no loss in efficiency of the substrate.

[0018] It is however also envisaged that the coagulant may be a salt or poly salt, preferably having a valency of between 1 to 4.

[0019] The metal nanoparticles may be formed from one or more metals selected from a group consisting of gold, silver, copper or titanium dioxide. The use of nanoparticles of other metals is however also envisaged. The individual said nanoparticles may have an average diameter of between 10 and 100 nm. The nanoparticles may preferably be agglomerated in aggregates having an average diameter of between 1 .1 to 10 or preferably 1 .1 to 7, or more preferably 1 .1 to 5 times the average diameter of the individual nanoparticles. Furthermore, the nanoparticles may coat between 5% to 100%, and preferably 25% to 90% of the substrate surface. [0020] The metal nanoparticles may be functionalised with an antigen or an antibody. The metal nanoparticles may be functionalised through a self assembling system such as thiol coupling or a bioaffinity system such as steptavidin-biotin type.

[0021 ] It is envisaged that the substrate may have a Raman enhancement factor (EF) of 1 ,000, 10,000, 100,000, 1 ,000,000 or higher.

[0022] According to another aspect of the present invention, there is provided a method of producing a metal nanoparticles treated cellulosic substrate for use as a surface enhanced Raman scattering (SERS) biodiagnostic platform including the steps of: a) adsorbing metal nanoparticles on the substrate from a nanoparticles suspension solution; and b) treating the nanoparticles with a coagulant so that the nanoparticles agglomerate on the substrate.

[0023] The coagulant may be a polymer that is adsorbed on the substrate from a polymer solution. The polymer may be added as a surface treatment on the substrate. Alternatively, the polymer may be added to the solution containing the metal nanoparticles suspension. As discussed earlier, the coagulant may alternatively be a salt or poly salt, preferably having a valency of between 1 to 4.

[0024] Paper or other cellulosic substrates treated with agglomerated metal nanoparticles provides a low-cost flexible platform for bioassays using SERS technology to increase the sensitivity (signal intensity) by more than 4 orders of magnitude. This paper can be used for health, environmental (water test), industrial and analytical applications. The metal nanoparticles can be

functionalized with any selective ligand sensitive to the analyte of interest using 2 convenient chemistries. The paper can be used as follows. The metal nanoparticle paper is first functionalized with a captor selective to the analyte of interest. This can be an antigen to detect an antibody, an enzyme to reveal a substrate or a ligand for an analyte. The functionalized metal nanoparticle paper is then contacted with the liquid sample of interest: bio-fluid (blood, urine, saliva, feces, water) or water- to adsorb the analyte of interest. Selectivity is insured by bio-recognition (enzyme-substrate, antibody-antigen) or specific ligand chemistry. The paper is then used, wet or dry, in a hand held battery operated or with a laboratory Raman scattering Spectrophotometer that will produce an intensity- wavelength spectrum. The peaks frequency is a function of the chemistry on the nanoparticles with analytes, and the intensity function of the concentration.

Selectivity is insured in 2 ways: with the ligand only capturing the target molecule of interest and with the Raman spectra specific to each substance.

[0025] The applicants discovered that the SERS intensity signal is directly proportional to metal nanoparticle surface coverage on paper and increases faster than linearly with the average size of nanoparticle aggregate. The heart of the invention lies in pre-treating paper with a polymer or other coagulant to engineer the exact nanoparticle surface coverage and average size aggregates. The effect of polymer concentration, charge density and molecular weight was quantified using a CPAM. Best performances were achieved with the high charge, high molecular weight CPAM used at higher concentration. The distribution of the nanoparticles through the thickness of paper also affects SERS and can be engineered. Best results were achieved with nanoparticles distributed over a thin interphase of a few paper fibers thickness.

[0026] The immobilization of biomolecules, such as antibodies and antigens, onto metal nanoparticles provides a good diagnostic platform in health and medicine. Taking advantage of the high affinity association between the

Streptomyces aw^'d7/7-derived protein, streptavidin, and biotin, the metal nanoparticle surface can be functionalized with the desired biomolecule.

Streptavidin has four active binding sites, thereby providing a stable and selective host protein for surface immobilization. Firstly, streptavidin is bound to the metal nanoparticle surface using biotinylated-thiol. Subsequently the desired biotinylated-biomolecule is bound to streptavidin, thus creating a biomolecular monolayer on the metal nanoparticle surface. The interaction of the biomolecule with a specific analyte can then be detected using SERS. No blockage is required to prevent competitive adsorption (such as with BSA, casein, milk as for ELISA tests). The metal nanoparticles can also be functionalized with the molecule of interested end capped with a thiol group; the molecule-thiol self assembles on gold/metal surfaces.

[0027] The invention has advantages over existing known technology for a few reasons. First it relies on simple contact method of the paper or other cellulosic substrate with a suspension of nanoparticle and does not require the electron beam lithography, focused ion beam patterning and thermal evaporation typical of nanofabrication. Second, the metal nanoparticles are adsorbed onto paper or other cellulosic substrate at high surface coverage and as aggregates of controlled size. SERS enhancement factor is known to increase exponentially with the number of nanoparticle-nanoparticle contact. Third, direct assembly of nanoparticles is performed instead of in-situ assembly. This allows a much better control of particle size, distribution in the substrate and reduces possibilities of contamination. Fourth, a polymer may be used to retain nanoparticles on the substrate. As bioactive papers are used WET, it is critical to retain the particles on paper during the test.

[0028] The previously noted prior art does not disclose the control of the size of nanoparticle aggregate or nanoparticle surface coverage on paper or other cellulosic substrate by coagulation of the nanoparticles. Coagulation is

significantly cheaper and versatile than the alternative technics of

nanofabrication. Coagulation can be engineered either by pre-treating the substrate with a polymer (bulk treatment or surface treatments: printing, spraying, surface sizing, coating) or by controlling the stability of the nanoparticle with salt (mono, bi, tri and 4 valency salt) or polymer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] It will be convenient to describe the invention with reference to the following examples and accompanying drawings. The particularity of the examples and drawings are not to be understood as superseding the generality of the preceding description of the invention.

[0030] In the drawings:

[0031 ] Figure 1 shows FESEM images and histograms of particle size distributions of nanoparticles showing the effect of different AuNP solutions;

[0032] Figure 2 shows a Raman spectrum and SERS intensity showing the effect of different AuNP solutions;

[0033] Figure 3 shows FESEM images and histograms of particle size distributions of nanoparticles showing the effect of different CPAM

concentrations;

[0034] Figure 4 respectively shows a Raman spectrum and a graph showing the relationship between the average particle size and surface coverage of the nanoparticles and the Raman Enhancement Factor (EF) of 4-ATP showing the effect of different CPAM concentrations;

[0035] Figure 5 shows FESEM images and histograms of particle size distribution of nanoparticles showing the effect of different polymer charge density;

[0036] Figure 6 respectively shows a Raman spectrum and a graph showing the relationship between the average particle size and the surface coverage of nanoparticles and the Raman Enhancement Factor (EF) of 4-ATP showing the effect of different polymer charge density; [0037] Figure 7 shows FESEM images and histograms of particle size distribution of nanoparticles showing the effect of different polymer molecular weight; and

[0038] Figure 8 respectively shows a Raman spectrum and a graph showing the relationship between the average particle size and surface coverage of nanoparticles and the Raman Enhancement Factor (EF) of 4-ATP showing the effect of different polymer molecular weight.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The invention will now be described with reference to the following four experimental test examples.

1 ) Effect of AuNp concentration

[0040] The first experimental example shows that the normal treatment of paper with AuNP solutions results in the adsorption of the AuNP as individual particles. The surface coverage is directly proportional to the concentration of the AuNP suspension with which is treated the paper. A surface coverage of AuNPs ranging from 1 .8 to 22.1 % was achieved on paper; however, the particles on the surfaces represented only 40% of the AuNPs' loading, with the majority uniformly adsorbed within the bulk of the paper. The SERS enhancement factor (EF) is directly proportional to the density of AUNPs on the paper. The adsorption of AuNPs on the surface and into the bulk of the paper presented a 3D multilayer architecture for intralayer and interlayer plasmon coupling. The z-distribution of AuNPs throughout the multilayer of cellulose fibers is believed to be responsible for amplifying the SERS signal via interlayer enhancement. METHOD

SYNTHESIS AND DEPOSITION OF NANOPARTICLES ON PAPER

[0041 ] AuNPs were synthesized by using 1 mM HAuCI₄.3H₂0 and 1 % aqueous Na₃C6H₅07.2H20 according to the Turkevich method [Turkevich, J., P.C. Stevenson, and J. Hillier, A study of the nucleation and growth processes in the synthesis of colloidal gold. Discussions of the Faraday Society 1 951 . 1 1 : p. 55- 75]. Filter papers were used as received and dipped into Petri dishes which contained 1 0 ml_ solution of AuNPs for 24 hours. After dipping, the paper substrates were rinsed thoroughly with distilled water to remove loosely bound AuNPs, and the papers were dried and equilibrated at 50% relative humidity and 23°C before further analysis.

PREPARATION OF RAMAN ACTIVE SUBSTRATES

[0042] Solutions of 1 mM of 4-ATP were prepared in ethanol. Since 4-ATP is well known for its strong affinity to the surface of AuNPs (its S-H bond is easily cleaved to form an Au-S bond upon adsorption), the dried AuNPs-deposited substrates were dipped into 2 ml_ of the 4-ATP ethanol solution for a shorter period of time of 5 minutes to create a 4-ATP monolayer on the substrates. After thorough rinsing with ethanol and drying, the treated papers were subjected to Raman characterization. The Raman enhancement factors (EF) of 1 mM of 4- ATP on a substrate was calculated according to the following equation:

where I_SERS is the intensity of a specific band in the SERS spectrum of 4-ATP and Ibuik is the intensity of the same band in the Raman spectrum from the bulk solution sample. For all spectra, the intensity of the band at 1 077 cm^"1 was used to calculate EF values. N_buik is the number of molecules of the bulk 4-ATP in the laser illumination volume while N_ads is the number of molecules adsorbed and sampled on the SERS active substrate within the laser spot.

INSTRUMENTATION

[0043] Field Emission Scanning Electron Microscopy (FESEM), which produces higher resolution, less sample charging and less damaged images than conventional FESEM, was performed using a JEOL 7001 Field Emission Gun (FEG) system operating at 5 kV and 180 pA. The Zeta potential and Dynamic Light Scattering (DLS) measurements were performed with a Zetasizer Nano ZS (Malvern Instruments) in a Folded Capillary cell (DTS1060) at 25 °C. UV-Vis absorbance was measured using a Varian Cary 300Bio spectrophotometer. All Raman and SERS spectra were obtained in air using a Renishaw Invia Raman microscope equipped with a 300 mW 633 nm laser. The laser beam was positioned through a Leica imaging microscope objective lens (50χ), whilst the instrument's wavenumber was calibrated with a silicon standard centered at 520.5 cm^"1 shift. Due to the smaller spot size of the laser compared with the large surface area of the samples, the spectra were obtained at different points of the surface. The position of the spectra bands remained the same, but differed only in intensity. The average Raman intensity of 5 measurements was presented after baseline subtraction from the control sample.

[0044] Figure 1 shows FESEM images and histograms of particle size distribution of filter papers dipped into (a) 0.02 mg/mL, (b) 0.05 mg/mL, (c) 0.10 mg/mL, (d) 0.1 5 mg/mL and (e) 0.20 mg/mL of AuNP solutions

[0045] Figure 2 shows (Left) Raman spectrum of 1 mM of 4-ATP adsorb on (a) plain filter paper and SERS spectra of 4-ATP on filter paper dipped in AuNP solutions of (b) 0.02 mg/mL, (c) 0.05 mg/mL, (d) 0.1 0 mg/mL, (e) 0.1 5 mg/mL and (f) 0.20 mg/mL. (Right) SERS intensity of 4-ATP at 1077 cm^"1 band from paper dipped in different concentration of AuNP solutions. 2) EFFECT OF POLYMER SOLUTION CONCENTRATION

[0046] In a second experimental example, paper is treated with polymer solutions of different concentrations. This example shows the drastic effect of controlled coagulation of the AuNP with a polymer. Paper was treated with a CPAM polymer solution (CPAM, 13 MD, 40% charge density); AuNP deposits as aggregates on paper and the average size of the aggregate increases with polymer concentration. SERS enhancement factor increases both with AuNP surface coverage and average particle size.

METHOD

DEPOSITION OF NANOPARTICLES ON PAPER

[0047] The method is similar to the first example except for the additional treatment of the paper with CPAM:

[0048] Hydrogen tetrachloroaurate trihydrate (HAuCI₄.3H₂0), sodium citrate tribasic dihydrate (Na₃C₆H₅0₇.2H₂0) and 4-aminothiophenol (4-ATP) were purchased from Sigma-Aldrich and used as received. The cationic polyacrylamide (CPAM) polymers were supplied by AQUA+TECH Switzerland from their SnowFlake Cationics product range, and used as received. These were copolymers of uncharged acrylamide with cationic dimethylaminoethylacrylate methyl chloride and identified as: 11 (5 wt% charge density, molecular weight 1 3 MDa), H1 (10 wt% charge density, molecular weight 1 3 MDa), F1 (40 wt% charge density, molecular weight 13 MDa) and F3 (40 wt % charge density, molecular weight 6 MDa). Whatman qualitative filter paper #1 , which consists of 98% a- cellulose, was selected as the paper substrate. Ultrapure water purified with a Millipore system (18 MQ.cm) was used in all aqueous solutions and rinsing procedures. [0049] Figure 3 shows FESEM images and histograms of particle size distribution of filter paper dipped in 1 .65 nM of AuNP solutions (a) without CPAM pre-treatment and with CPAM pre-treatment of (b) 0.01 mg/ml (c) 0.05 mg/ml and (d) 0.10 mg/ml polymer concentration.

[0050] Figure 4 shows (Left) Raman spectrum of 4-ATP adsorb on (a) plain filter paper, SERS spectra of 4-ATP on AuNPs paper (b) without CPAM pre- treatment and with CPAM pre-treatment of (c) 0.01 mg/ml (d) 0.05 mg/ml and (e) 0.10 mg/ml polymer's concentration. (Right) Relationship between the average size, surface coverage of AuNP aggregates and the EF of 4-ATP measured at the 1077 cm-1 band.

3) EFFECT OF POLYMER CHARGE DENSITY

[0051 ] In a third experimental example, paper is treated with polymer solutions of different charge density (constant molecular weight and polymer

concentration). AuNP aggregate size and surface coverage on paper both increases with polymer charge density. SERS enhancement factor increases both with AuNP surface coverage and average particle size.

METHOD

[0052] The method is the same as described in the second example except for the treatment of the paper with polymer of different charge density.

[0053] Figure 5 shows FESEM images and histograms of particle size distribution of AuNPs-CPAM papers with polymer's charge density of (a) 5 wt%, (b) 10 wt% and (c) 40wt%.

[0054] Figure 6 shows (Left) SERS spectra of 4-ATP on AuNPs-CPAM papers with polymer's charge density of (a) 5 wt%, (b) 10 wt% and (c) 40wt%. (Right) Relationship between the average size and surface coverage of AuNP

aggregates and the EF of 4-ATP measured at the 1077 cm-1 band.

4) EFFECT OF POLYMER MOLECULAR WEIGHT

[0055] In a fourth experimental example, paper is treated with polymer solutions of different molecular weight (constant charge density and polymer concentration). AuNP aggregate size and surface coverage on paper both increases with polymer molecular weight. SERS enhancement factor increases both with AUNP surface coverage and average particle size.

METHOD

[0056] The method is the same as described in the second example except for the treatment of the paper with polymer of different molecular weight.

[0057] Figure 7 shows FESEM images and histograms of particle size distribution of AuNPs-CPAM papers with polymer's molecular weight of (a) 6 MDa and (b) 13 MDa.

[0058] Figure 8 shows (Left) SERS spectra of 4-ATP on AuNPs-CPAM papers with polymer's molecular weight of (a) 6 MDa and (b) 13 MDa. (Right)

Relationship between the average size and surface coverage of AuNP

aggregates and the EF of 4-ATP measured at the 1077 cm-1 band.

[0059] Modifications and variations as would be deemed obvious to the person skilled in the art are included within the ambit of the present invention as claimed in the appended claims.

Claims

CLAIMS:

1 . A metal nanoparticle treated cellulosic substrate for use as a surface enhanced Raman scattering (SERS) biodiagnostic platform, the substrate including metal nanoparticles coating the substrate, wherein the nanoparticles are agglomerated on the substrate due to pre-treatment with a coagulant.

2. A metal nanoparticle treated cellulosic substrate according to claim 1 , wherein the substrate is formed from paper.

3. A metal nanoparticle treated cellulosic substrate according to claim 2, wherein the paper has a basic weight between 15 to 500 g/m², and preferably 40 to 400 g/m².

4. A metal nanoparticle treated cellulosic substrate according to claims 1 or 2, wherein the coagulant is a polymer.

5. A metal nanoparticle treated cellulosic substrate according to claim 4, wherein the polymer has a molecular weight of between 100,000 Dalton to 20,000,000 Dalton.

6. A metal nanoparticle treated cellulosic substrate according to claim 4, wherein the polymer has a charge density of between 5% to 100% based on monomer charge.

7. A metal nanoparticle treated cellulosic substrate according to claim 4, wherein the polymer has a linear, branched, dendritic, alternating or block morphology.

8. A metal nanoparticle treated cellulosic substrate according to claim 4, wherein the polymer is selected from a group consisting of CPAM, PEI, PEO, PVAM, PAE, polyDADMAC and copolymers and blends thereof.

9. A metal nanoparticle treated cellulosic substrate according to claim 1 , wherein the coagulant is a salt or poly salt, preferably having a valency of between 1 to 4.

10. A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein the metal nanoparticles are formed from one or more metals selected from a group consisting of gold, silver, copper or titanium dioxide.

1 1 . A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein individual said nanoparticles have an average diameter of between 10 and 100 nm.

12. A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein the nanoparticles are agglomerated in aggregates having an average diameter of between 1 .1 to 1 0 or preferably 1 .1 to 7, or more preferably 1 .1 to 5 times the average diameter of the individual nanoparticles.

13. A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein the nanoparticles coat between 5% to 100%, and preferably 25% to 90% of a surface of the substrate.

14. A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein the metal nanoparticles are functionalised with an antigen or an antibody.

15. A metal nanoparticle treated cellulosic substrate according to any one of claims 1 to 13, wherein the metal nanoparticles are functionalised through a self assembling system such as thiol coupling or a bioaffinity system such as steptavidin-biotin type.

16. A metal nanoparticle treated cellulosic substrate according to any one of the preceding claims, wherein the substrate has a Raman enhancement factor (EF) of 1 ,000, 10,000, 100,000, 1 ,000,000 or higher.

17. A method of producing a metal nanoparticles treated cellulosic substrate for use as a surface enhanced Raman scattering (SERS) biodiagnostic platform including the steps of: a) adsorbing metal nanoparticles on the substrate from a nanoparticles suspension; and b) treating the nanoparticles with a coagulant so that the nanoparticles agglomerate on the substrate.

18. A method of producing a metal nanoparticle treated cellulosic substrate according to claim 17, wherein the coagulant is a polymer that is adsorbed on the substrate from a polymer solution.

19. A method of producing a metal nanoparticle treated cellulosic substrate according to claim 17, wherein the coagulant is a polymer that is added as a surface treatment on the substrate.

20. A method of producing a metal nanoparticle treated cellulosic substrate according to claim 17, wherein the coagulant is a polymer that is added to the nanoparticles suspension.

21 . A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 18 to 20, wherein the polymer has a molecular weight of between 100,000 Dalton to 20,000,000 Dalton.

22. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 18 to 20, wherein the polymer has a charge density of between 5% to 100% based on monomer charge.

23. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 18 to 20, wherein the polymer has a linear, branched, dendritic, alternating or block morphology.

24. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 18 to 20, wherein the polymer is selected from a group consisting of CPA , PEI, PEO, PVAM, PAE, polyDADMAC and copolymers and blends thereof.

25. A method of producing a metal nanoparticle treated cellulosic substrate according to claim 17, wherein the coagulant is a salt or poly salt, preferably having a valency of between 1 to 4.

26. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 25, wherein the substrate is formed from paper.

27. A method of producing a metal nanoparticle treated cellulosic substrate according to claim 26, wherein the paper has a basic weight between 15 to 500 g/m², and preferably 40 to 400 g/m².

28. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 27, wherein the metal nanoparticles are formed from one or more metals selected from a group consisting of gold, silver, copper or titanium dioxide.

29. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 28, wherein individual said nanoparticles have an average diameter of between 10 and 100 nm.

30. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 29, wherein the nanoparticles are agglomerated in aggregates having an average diameter of between 1 .1 to 10 or preferably 1 .1 to 7, or more preferably 1 .1 to 5 times the average diameter of the individual nanoparticles.

31 . A method of producing a metal nanoparticle treated cellulosic substrate according to any one claims 17 to 30, wherein the nanoparticles coat between 5% to 100%, and preferably 25% to 90% of a surface of the substrate.

32. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 30, wherein the metal nanoparticles are functionalised with an antigen or an antibody.

33. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 32, wherein the metal nanoparticles are functionalised through a self assembling system such as thiol coupling or a bioaffinity system such as steptavidin-biotin type.

34. A method of producing a metal nanoparticle treated cellulosic substrate according to any one of claims 17 to 33, wherein the substrate has a Raman enhancement factor (EF) of 1 ,000, 10,000, 100,000, 1 ,000,000 or higher.