Separation of minerals using short peptides or mimetics thereof

Abstract

The disclosure relates to the separation or recovery of desired mineral(s) from various materials such as, for example, ore, e-waste and industrial wastewater, involving the use of a short peptide or mimetic thereof which selectively binds to one or more mineral(s).

Description

SEPARATION OF MINERALS USING SHORT PEPTIDES OR MIMETICS THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to the separation or recovery of desired mineral(s) from various materials such as, for example, ore, e-waste and industrial wastewater, involving the use of a short peptide or mimetic thereof which selectively binds to one or more miner al(s).PRIORITY DOCUMENT

[0002] The present application claims priority from Australian Provisional Patent Application No. 2023902544 titled "SEPARATION OF MINERALS" and filed on 10 August 2023, the content of which is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Valuable minerals play essential roles in our modern society. The urbanisation of the developing world, the emerging green economy to rebuild our energy systems, and the digital revolution have resulted in an unprecedented global demand for metals. For example, low carbon technologies require metals such as lithium, cobalt, nickel, manganese and copper, while smart-phones contain up to 62 different metals including the rare earths. With a projected global population of nine billion by 2050, demographics forecasting indicates strong increases in the demand for these resources over the long term. However, the declining grade of the more accessible ores and increasing complexities of the mineralogy, can make current mineral processing methodologies technologically, economically and environmentally unsustainable. Therefore, developing new technologies, which may make use of new reagents, for the more effective and more sustainable separation and recovery of minerals, is urgently needed.

[0004] In work leading to the present disclosure, the Applicant(s) developed a phage display-based method for screening short peptides for their potential to bind to a target desired mineral(s). This work led to the identification of a group of heptapeptides ("7-mers") with surprisingly specific mineral binding capabilities, which may enable, for example, a relatively simple and efficient means for the separation of valuable minerals from ore preparations, tailings or waste / recycled materials (eg electronics waste ("e-waste") and waste from green / renewable energy infrastructure).SUMMARY

[0005] According to a first aspect, the present disclosure provides a method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.

[0006] The method may be conducted with any suitable material such as an ore, ore preparation, tailings, tailings preparations, tailings dam water and / or settled materials from the bottom of a tailings dam, contaminated soils, wastewater, and waste / recycled materials, and may be applied to the separation of at least one desired mineral selected from, for example, silver, gold, silica, magnetite, hematite or a rare earth element (REE), particularly neodymium and dysprosium.

[0007] In a second aspect, the disclosure provides a peptide comprising an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36, or a mimetic thereof.

[0008] The peptide or mimetic thereof may also be of use in the analysis of the mineral content of a material, especially an ore sample or ore preparation (ie in a mineral diagnosis method).

[0009] Thus, in a third aspect, the present disclosure provides a method of assaying a material for the presence of at least one mineral, wherein the method comprises contacting the material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, determining peptide or mimetic thereof that is bound to the desired mineral, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.BRIEF DESCRIPTION OF FIGURES

[0010] Figure 1 provides a schematic representation of the phage display method used to screen for mineral-binding peptides (with silver powder);

[0011] Figure 2 provides the results of binding affinity analysis of the Agl peptide as determined by isothermal titration calorimetry (ITC): a. 50 pM AgNO? in sample cell and 500 M Agl peptide in syringe, b. 50 pM AgNO? in sample cell and 25 mM HEPES in syringe, c. 25 mM HEPES in sample cell and 500 pM Agl peptide in syringe, and d. 50 pM NaNO? in sample cell and 500 pM Agl peptide in syringe;

[0012] Figure 3 provides results indicating the effect of different parameters on binding affinity and stoichiometry (Ag+per Agl) between Ag+and the Agl peptide: a and b. Temperature effect; c and d. pH effect; and e and f. salt concentration effect;

[0013] Figure 4 provides the chemical structure of the Agl peptide, and the variants and control peptides described in the example(s);

[0014] Figure 5 provides the raw data, binding isotherms, and thermodynamics information obtained from ITC over 19 injections of the Agl peptide, and the variants and control peptides into AgNCL: a. no peptide, b. Agl, .c. Agl-GGGS, d. Agl-2F, e. Agl-Agl, f. Agl-GGGS-Agl, g. 7G, h. GGGS (SEQ ID NO: 37), i. binding constant KA, j. binding stoichiometry (Ag+ / Agl) N, k. enthalpy change AH, 1. entropy change -TAS, and m. Gibbs free energy change AG;

[0015] Figure 6 provides results of experiments to assess immediate size, absorbance and morphology change of Ag nanoparticles (NPs) upon mixing with the Agl peptide, and the variants and control peptides: a. Z- Average change determined by dynamic light scattering (DLS), b. absorbance change determined by UV-Vis spectroscopy, and c. morphology change determined by transmission electron microscopy (TEM);

[0016] Figure 7 provides the results of experimentation showing selective precipitation of Ag NPs over SiOz NPs by Agl and Agl peptide variants from a mixture of Ag NPs and SiO NPs: a. NP size, b. PDI, and c. Zeta-Potential (c) of Ag NPs and SiO NPs determined by DLS. Photographs, quantitative precipitation analysis of Ag NPs, quantitative precipitation analysis of Si CL NPs, and separation factor (Ag / Si) after 16 h (d, e, f, g) and 24 h (h, i, j, k) are also provided;

[0017] Figure 8 provides graphical results showing the size and PDI change of Ag NPs, SiCL NPs, and Ag NPs+SiCL NPs caused by various peptides over 24 h. a. no peptide, b. Agl, c. Agl-GGGS, d. Agl-2F, e. Agl-Agl, f. Agl-GGGS-Agl, g. 7G, and h. GGGS (SEQ ID NO: 37)

[0018] Figure 9 provides results obtained using the Agl peptide variant, AMI -Agl. a. ITC thermogram of AMI -Agl binding to AgNOs, Kd (b, c) and N I for the binding of Agl and AMI -Agl to Ag+. Distribution of Ag NPs (d) and SiO2 NPs (e) post-emulsion destabilization, f. Quantification of the separation factor illustrating the efficiency of AMI -Agl in selectively separating Ag NPs from SiO2 NPs. Data are shown as mean ± s.d. The P value was determined by using the unpaired t-test;

[0019] Figure 10 provides graphical results showing the performance of further Agl peptide variants, this time including sequences from elastin-like proteins (ELPs). Distribution of Ag NPs (a) and SiO2 NPs (b) in suspension and as precipitates after treatment with ELP-Agl and ELP-Agl-GGS-Agl for4 h at 22 and 40°C. Separation factors (Ag / Si) achieved for Ag NP and Si02 NP mixtures at 4 (c) and 24 h (d);

[0020] Figure 11 provides a schematic diagram of the recycling process for ELP-based proteins; and

[0021] Figure 12 provides results showing: a. the recovery rates of recycled ELP-Agl and ELP-Agl- GGS-Agl; and b. Quantification of Ag NP distribution post-treatment with fresh and recycled ELP peptides, showing efficient precipitation within 4 h. Data are shown as mean ± s.d.DETAILED DESCRIPTION

[0022] The Applicant(s) developed a phage display-based method for screening short random 7-mer peptides for their potential to bind to a target mineral(s). This work led to the identification of a group of heptapeptides with surprisingly specific mineral binding capabilities, and which may enable, for example, a relatively simple and efficient means for the separation of valuable minerals from ore preparations, tailings or waste / recycled materials. For instance, in the example(s) hereinafter, peptides were identified that specifically or selectively bind to silver particles and were shown to be capable of separating the silver particles from a model waste material (ie silica particles) with an exceptionally high separation factor (>1400).

[0023] In a first aspect, the present disclosure provides a method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material (eg a feedstock) in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.

[0024] The method may be conducted with any suitable material, such as a feedstock, comprising the at least one desired mineral. Examples of suitable materials include an ore, ore preparation (eg an ore that may have been, for example, crushed or otherwise comminuted, sieved (ie "sized") to comprise particles of a desired size, concentrated using any of the standard methodologies known to those skilled in the art such as gravity concentration and froth flotation (ie such that the ore preparation is what is typically referred to as the "concentrates"), and / or leaching so as to convert the mineral(s) into a form more conducive to separation by the method of the disclosure (eg by leaching the desired mineral(s) from a solid material such as an ore to form, for example, a soluble salt)), tailings (which include residual materials of mineral processing (residues) which may comprise minerals whose extraction may be regarded as uneconomic by current mineral processing methodologies, but which could potentially be recovered using the separation method of the disclosure), tailings preparations (egtailings that may have been, for example, crushed or otherwise comminuted, sieved, concentrated, and / or subjected to a leaching step) and tailings dam water and / or settled materials from the bottom of a tailings dam. Other examples of suitable materials include contaminated soils (eg for remediation), wastewater (eg for treatment of industrial wastewater), and waste / recycled materials (eg electronics waste ("e-waste") and waste from green / renewable energy infrastructure such as old solar panels / cells, wind turbine blades, and spent batteries and storage batteries). Typically, these waste / recycled materials will be prepared for use in the separation method of the disclosure by, for example, crushing and / or the application of other techniques for comminuting the materials, sieving (eg to achieve a material with a maximum particle size), concentration of components and / or particles within the material which comprise the desired miner al(s), and / or by subjecting the material to a leaching step so that the desired mineral(s) within the material is converted to a form such as, for example, a soluble salt which may be more readily separable.

[0025] The method of the first aspect requires that the material be provided in a fluid form; preferably as a liquid. Some suitable materials such as wastewater and tailings dam water may be immediately suitable for use in the method of the disclosure, whereas in other cases, the material may need to be rendered into, for example, a suitable liquid solution or suspension. For example, materials which have, for example, been comminuted and sieved into a fine particulate preparation (eg comprising particles in the range of 10-500 pm) may be added to a solvent (eg water, organic solvent, inorganic solvent or ionic liquid) or liquid carrier so as to provide, for example, a slurry for use in the method of the disclosure. The fluid form of the material may comprise the desired mineral(s) in, for example, a more readily separable (extractable) form such as a compound or soluble salt. Otherwise, the desired mineral(s) may be present in the usual mineral form or in an elemental form (eg particles, particularly nanoparticles, of an elemental mineral such as silver or gold). In some embodiments, the fluid form of the material may comprise the desired mineral(s) in two or more forms (eg comprising mineral(s) in both an elemental and salt form).

[0026] The material in fluid form may be contacted with the peptide or mimetic thereof in any suitable manner including those which will be readily apparent to those skilled in the art, so as to enable the peptide or mimetic thereof to selectively bind to the desired mineral(s) contained in the material, such that the mineral(s) bound to the peptide or mimetic thereof may be separated from the (residual) fluid material. As used herein, the term "selectively binds", and variants thereof, means that the peptide or mimetic thereof binds with substantially greater affinity to the target desired mineral(s) than it does to other (unwanted) substances present in the material, so as to enable the desired mineral(s) as bound to the peptide or mimetic thereof to be separable from the unwanted substances. Preferably, a peptide or mimetic thereof which at least selectively binds to the desired mineral(s) does so with binding constant KAof >1* 104, more preferably >1* 105, and most preferably >5* 105(under conditions of room temperature, pH 5-7 and about 5% salt).

[0027] In some embodiments, the peptide or mimetic thereof may be provided on the surface of a solid support or substrate such as, for example, on the surface of a filter membrane or sieve, and / or as provided on the surface of suitable beads (eg inert beads such as ceramic beads or polymeric beads) which may be provided in a separation column. In such embodiments, the fluid material may be passed through the filter membrane, sieve and / or column under conditions suitable for the binding of the peptide or mimetic thereof to the desired mineral(s) such that the desired mineral(s) are "captured" and thereafter recovered from the respective solid supports by any treatment and / or process to release the mineral(s) from the peptide or mimetic thereof (eg an elution step such as, for example, by changing the pH with an acid or alkali flush, or by flushing with a salt solution to form a salt of the desired mineral). In some other embodiments, the peptide or mimetic thereof may be provided on the surface of bubbles of a suitable gas (preferably air) formed by, for example, bubbling of the gas through the fluid material such as in a flotation separation process such as any of those well known to those skilled in the art (eg froth flotation). In embodiments comprising a flotation separation process, the fluid for the material may comprise water as the solvent or carrier, and the peptide or mimetic thereof may be selected on the basis of hydrophobicity (ie the peptide or mimetic thereof may be relatively hydrophobic as may be predicted / determined using well known hydrophocity algorithms such as that described in Fauchere JL and V Pliska. Eur J Med Chem 10:369, 1983) and / or modified so as to be relatively hydrophobic, so that the hydrophobic peptide or mimetic thereof assemble at the interface between the gaseous bubbles and the water solvent or carrier. In this way, mineral(s) which become bound to the peptide or mimetic thereof are effectively carried by the bubbles to the surface (or top) of the chamber or cell within which the flotation separation is conducted, and from where the bubbles (froth) and bound mineral(s) may be recovered or removed. The peptide or mimetic thereof may be, for example, conveniently provided in the gas (eg air) forming the bubbles within the flotation separation chamber or cell.

[0028] As will be appreciated by those skilled in the art, the conditions under which the fluid material is contacted with the peptide or mimetic thereof may be adjusted so as to optimise the binding of the peptide or mimetic thereof with the desired mineral(s) contained in the material. In some cases, sub- optimal conditions may be employed for economic reasons (eg to reduce energy input etc). However, typically, the conditions used will include: a temperature in the range of 4 to 45 °C, more preferably 10 to 35 °C, and most preferably 15 to 30 °C; and a pH in the range of 4 to 8, but more preferably 5 to 7. In some embodiments, the conditions under which the fluid material is contacted with the peptide or mimetic thereof may comprise a salt concentration in the range of 0.1 to 10 wt% (based upon the total weight of the fluid material).

[0029] Techniques for modifying a peptide or mimetic thereof so as to be hydrophobic may include, for example, one or more amino acid substitution or addition so as to introduce one or more hydrophobic amino acid(s), preferably one or more hydrophobic amino acid(s) selected fromphenylalanine (F), leucine (L), isoleucine (I), tyrosine (Y), tryptophan (W), valine (V), methionine (M) and proline (P). In some particular embodiments, the peptide or mimetic thereof may comprise the addition of at least one phenylalanine residue (eg the addition of an FF dipeptide motif to the N- terminal but more preferably, C-terminal of a mineral -binding peptide or mimetic). Other suitable techniques for modifying the peptide or mimetic thereof may include the addition of a short hydrophobic peptide sequence (eg to the N- and / or C-terminal, but more preferably, to the N-terminal, preferably of 2 to 25 amino acids in length or more preferably of about 4 to 21 amino acids in length (eg AAIV (SEQ ID NO: 45), VVLGAAIV (SEQ ID NO: 46) and MKQLADS-LHQLARQ- VSRLEHA; SEQ ID NO: 53), and / or a hydrophobic moiety. For example, a hydrophobic moiety such as a long hydrocarbon chain (eg a compound comprising a hydrocarbon chain with 5 to 30 carbon atoms (C5-C30 chain), or fatty acid (eg a fatty acid with a hydrocarbon chain of 10 to 30 carbon atoms (C10-C30)), or a suitable hydrophobic polymer (eg a polymer selected from the group consisting of polyethylene, polystyrene, polyvinylchloride, polytetrafluorethylene, polyesters and polyurethanes).

[0030] The addition of the amino acid sequence: MKQLADS-LHQLARQ-VSRLEHA (SEQ ID NO: 53), known as the AMI peptide surfactant, represents a particularly preferred embodiment for modifying the peptide or mimetic thereof so as to be hydrophobic or more hydrophobic, thereby enabling or enhancing separation of minerals (ie as bound to the peptide or mimetic thereof) by a flotation separation process such as any of those well known to those skilled in the art (eg froth flotation). However, other peptide surfactants (otherwise referred to as "surfactant-like peptides" or "Pepfactants") as are well known to those skilled in the art may also be suitable, such as those described in the review by Li Y et al., Adv Funct Mater 33(7):2210387 (2022) and Li Y et al., Coord Chem Rev 213418 (2020).

[0031] The method of the first aspect may be applied to the separation of at least one desired mineral selected from, for example, precious metals and other metals such as lithium. However, as will be apparent from the above, the desired mineral(s) may also be separated in a non-elemental form such as, for example, a salt form. Thus, the method may also be applied to the separation of at least one desired mineral selected from, for example, precious metal ions and precious metal ion complexes. As used herein, the term "precious metal", and variants thereof, means one or more of gold, silver and the platinum group metals (ie platinum, palladium, rhodium, iridium, ruthenium and osmium, the term "precious metal ion" means an ion of a precious metal, and the term "precious metal ion complex" means an organic or inorganic metal ion complex comprising an ion of a precious metal. In some preferred embodiments, the method is applied to the separation of at least one mineral selected from the group consisting of silver, gold, lithium, silica, magnetite, hematite or a rare earth element, preferably neodymium and dysprosium, and any metal ion forms thereof (eg Ag+).

[0032] In some embodiments, the method is applied to the separation of at least silver and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of KAIHPMR (SEQ ID NO: 1), NSMKHVH (SEQ ID NO: 2), HPITRHK (SEQ ID NO: 3), and RPTKMHR (SEQ ID NO: 4), or a mimetic thereof. Preferably, in these embodiments, the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of the following amino acid sequence: KAIHPMR (SEQ ID NO: 1). The peptide consisting of SEQ ID NO: 1 is referred herein as the Agl peptide or more simply, Agl.

[0033] In some embodiments, the method is applied to the separation of at least gold and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of HSTAPQL (SEQ ID NO: 5), TGFPPYH (SEQ ID NO: 6), LYASRVP (SEQ ID NO: 7), GTGAYDA (SEQ ID NO: 8), KAQTLPA (SEQ ID NO: 9), FNNKHSS (SEQ ID NO: 10), LVTVHYS (SEQ ID NO: 11), TAIRDEL (SEQ ID NO: 12), NHLDDAK (SEQ ID NO: 13), LSLRPIP (SEQ ID NO: 14), SYVGHSP (SEQ ID NO: 15), LPTKPQL (SEQ ID NO: 16) and VAAREYS (SEQ ID NO: 17), or a mimetic thereof.

[0034] In some embodiments, the method is applied to the separation of at least silica (eg quartz) and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of HFKLPKK (SEQ ID NO: 18) and KPLKLPR (SEQ ID NO: 19), or a mimetic thereof.

[0035] In some embodiments, the method is applied to the separation of at least magnetite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of GSLGYTG (SEQ ID NO: 20) and WSLGYTG (SEQ ID NO: 21), or a mimetic thereof.

[0036] In some embodiments, the method is applied to the separation of at least hematite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), VAVPLHN (SEQ ID NO: 22), HSWRAPY (SEQ ID NO: 23), FNISSSR (SEQ ID NO: 24), NHAQFFK (SEQ ID NO: 25), and SHLIAER (SEQ ID NO: 26), HFHPLES (SEQ ID NO: 27), or a mimetic thereof.

[0037] In some embodiments, the method is applied to the separation of at least neodymium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), IYAKAPI (SEQ ID NO: 28), LPPYELH (SEQ ID NO: 29), HIGAAYE (SEQ ID NO: 30), and GSPYLFV (SEQ ID NO: 31), or a mimetic thereof.

[0038] In some embodiments, the method is applied to the separation of at least dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), HPSTWHK (SEQ ID NO: 32), FPVVTRN (SEQ ID NO: 33), QSFGPHP (SEQ ID NO: 34), and SYSLNPF (SEQ ID NO: 35), or a mimetic thereof.

[0039] In some embodiments, the method is applied to the separation of one or more of magnetite, hematite, neodymium and dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of the following amino acid sequence:(I) X2SLGYTG (SEQ ID NO: 36); where X2is any proteinaceous amino acid or is null, or more preferably is selected from glycine (G) / alanine (A) / valine (V) / leucine (L) / isoleucine (I) and tryptophan (W) / phenylalanine (F) / tyrosine (Y) or is null (ie is absent), or even more preferably is selected from G and W or is null. Exemplary peptides according to (I) include GSLGYTG (SEQ ID NO: 20) and WSLGYTG (SEQ ID NO: 21). As shown hereinafter, the peptide with the amino acid sequence shown as SEQ ID NO: 20 (referred herein as Magi) shows selectivity or specificity to magnetite. On the other hand, the peptide with the amino acid sequence shown as SEQ ID NO: 21 (referred herein as HemlMag2) shows selectivity to magnetite, hematite, neodymium and dysprosium, and is therefore capable of enabling the separation of each and all of these minerals (if present) from the fluid material.

[0040] In some embodiments, the method of the disclosure may be operated in a "multiplex" manner, whereby two or more desired mineral(s) present in the material are targeted for separation. Means for conducting such a method of separation will be readily apparent to those skilled in the art and may, for example, comprise the use of a solid support with two or more different zones, wherein each zone comprises a peptide or mimetic thereof which selectively binds to a different mineral. For example, a first zone on the solid support may comprise a peptide or mimetic thereof which selectively binds to silver, and a second zone on the solid support may comprise a peptide or mimetic thereof which selectively binds to gold. The different minerals (eg silver and gold) may subsequently be separately recovered from the respective first and second zones.

[0041] Techniques for modifying the peptide or mimetic thereof have been discussed above (eg so as to make the peptide or mimetic hydrophobic). Other modifications contemplated by the present disclosure include, for example, C-terminal amidation to remove the C-terminal negative charge, and modification to generate a peptide comprising more than one "copy" of the amino acid sequence (eg a dimeric or trimeric form), wherein the copies of the amino acid sequence is / are repeated in a head to tail arrangement such that the C-terminal residue of a first copy of the amino acid sequence is adjacent the N-terminal residue of the second copy of the amino acid sequence, or wherein more than two copies of the amino acid sequence are contemplated, the C-terminal residue of the second copy of the amino acid sequence is adjacent the N-terminal of a third copy of the amino acid sequence, and so on. However, it will also be apparent to those skilled in the art that the amino acid sequences may be joined by a short linker sequence or linker group that could potentially be used to link the copies of the amino acid sequence together. Suitable linker sequences are well known to those skilled in the art and include, for example, short amino acid sequences comprising 2-15 amino acids, preferably 5-10 amino acids, and which may be, optionally, solely or predominantly composed of "flexible" amino acids such as glycine (G) and serine (S) (eg well known "GS linkers") so that the copies of the amino acid sequence are free to move relative to one another. Particularly suitable linker sequences include the following: GGS and GGGS (SEQ ID NO: 37). Suitable linker groups may include, for example, polyethylene glycol (PEG)-based linkers and disulphide (-S-S-) linkers.

[0042] A further type of modification of the peptide or mimetic thereof contemplated by the present disclosure includes the addition of an FF dipeptide motif to the N-terminal but more preferably, C- terminal of a mineral-binding peptide or mimetic. As discussed above, the addition of an FF dipeptide motif may enable the method of the present disclosure to be conducted using a flotation separation process. However, in addition, and as is shown in the example(s) hereinafter, the addition of an FF dipeptide motif may also enhance the binding affinity of the peptide or mimetic for the desired miner al(s).

[0043] In some embodiments, the method of the first aspect preferably comprises the step of contacting the fluid material with a peptide which selectively binds to the desired mineral(s). However, in other embodiments, the method comprises the step of contacting the fluid material with a mimetic which selectively binds to the desired mineral(s), that is, a mimetic of a peptide comprising an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36. Suitable peptide mimetics may be designed using any of the methods well known to those skilled in the art for designing mimetics of peptides based upon amino acid sequences in the absence of secondary and tertiary structural information (eg as described in Kirschenbaum K et al., Curr Opin Struct Biol 9:530-535, 1999; the content of which is hereby incorporated by reference in its entirety). For example, peptide mimetics may be produced by substituting amino acid side chains with nonamino acid side chains (eg substituting aromatic residues of the peptides with other aryl groups) andsubstituting amino- and / or carboxy-termini with various substituents (eg substituting aliphatic groups to increase hydrophobicity). Alternatively, suitable peptide mimetics may be so-called peptoids (ie non-peptides) which include modification of the peptide backbone (ie introducing amide bond surrogates by, for example, replacing the nitrogen atoms in the backbone with carbon atoms), or which includes / V-substitutcd glycine residues, one or more D-amino acids (in place of L-amino acid(s)) and / or one or more a-amino acids (in place of P-amino acids or y-amino acids). Further, suitable mimetics of a peptide comprising an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36 include "retro-inverso peptides" where the peptide bonds are reversed and D-amino acids assembled in reverse order to the order of the L-amino acids in the amino acid sequence upon which the mimetic is based, and other non-peptide frameworks such as steroids, saccharides, benzazepine 1,3,4- trisubstituted pyrrolidinone, pyridones and pyridopyrazines.

[0044] The peptides or mimetics thereof may be produced using any of the methodologies known to those skilled in the art. For example, for the production of a peptide, the peptide may be produced by any of the standard protein synthesis methods (especially for a peptide of < 50 amino acids in length) or by recombinant techniques (especially where it is desired to produce the peptide as linked to, for example, a fusion protein) involving, for example, the introduction of a polynucleotide molecule encoding the particular peptide into a suitable host cell (eg a host cell selected from bacterial cells such as E. coli, Streptomyces and S. typhimuriunr, fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as Chinese hamster ovary (CHO), monkey kidney (COS) cells and human embryonic kidney 293 (HEK 293) cells; and plant cells) and culturing the cell under conditions suitable for the expression of the particular peptide. In some particular embodiments, the peptide may be expressed by recombinant techniques in the form of a fusion protein with a fusion partner located at the N- and / or C-terminal of the peptide. The fusion partner(s) may be linked to the peptide via a short linker sequence (eg 2 to 10 amino acids in length) as is well known to those skilled in the art. The linker sequence may be readily cleavable (eg by including a site for enzymatic cleavage) to "release" the peptide from the fusion partner. The fusion partner, as is also well known to those skilled in the art, may confer various additional functionalities or properties such as enhanced expression levels or enable improved peptide recovery. Examples of fusion partners that may be particularly suitable for expression with a peptide according to the present disclosure are elastin-like polypeptides (ELPs) (Kowalczyk T et al., World J Microbiol Biotechnol 30:2141-2152, 2014; the content of which is hereby incorporated by reference in its entirety), which enable easy purification of the peptide by forming readily isolated / separable aggregates at elevated temperature. As such, the ELP acts as a "purification tag" for the peptide (Hassouneh W et al., Curr Protoc Protein Sci Unit-6.11, 2010; the content of which is hereby incorporated by reference in its entirety). If the ELP is linked to the peptide by, for example, a cleavable linker sequence such as one that may be cleaved by an enzyme such as thrombin or TEV (tobacco etch virus) protease, then the peptide can besubsequently released from the ELP and prepared for use. However, in other embodiments, the peptide or mimetic thereof may be used in a method according to the present disclosure with the ELP remaining attached. For example, in a method for separating at least one desired mineral from material comprising same, the ELP-peptide / mimetic may be advantageously employed to allow for, for example, temperature-sensitive separation (resulting in the formation of readily isolated / separable aggregates), which may enable faster processing of the materials and mineral recovery with possibly higher separation factors. Further, once the mineral(s) have been recovered from the ELP- peptide / mimetic, the ELP-peptide / mimetic may be recycled for re-use.

[0045] Thus, in yet a further type of modification of the peptide or mimetic thereof contemplated by the present disclosure, the peptide or mimetic further comprises a peptide, oligopeptide or polypeptide sequence characterised by reversible solubility in response to temperature. In some preferred embodiments, the said peptide, oligopeptide or polypeptide sequence comprises the following pentapeptide sequence: VPGX'G (SEQ ID NO: 50), wherein X1is any proteinaceous amino acid except proline (Pro). Further, in some particularly preferred embodiments, the said peptide, oligopeptide or polypeptide sequence comprises multiple copy repeats of the pentapeptide (eg 2 to 100 repeats, or 5 to 75 repeats, or 10 to 75 repeats, or 25 to 75 repeats or about 50 to 60 repeats), preferably in a head to tail repeat arrangement. By way of example, the separation of at least silver, the peptide or mimetic thereof may have the following amino acid sequence: H6-SSGGVG-(VPGVG)s9- VGVPWP-(GS)7-KAIHPMR (SEQ ID NO: 48) or H6-SSGGVG-(VPGVG)59-VGVPWP-(GS)7- KAIHPMR-GGS-KAIHPMR (SEQ ID NO: 49).

[0046] In a second aspect, the disclosure provides a peptide comprising an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36, or a mimetic thereof.

[0047] In some embodiments, the peptide or mimetic thereof is a peptide which consists of an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36, or is a mimetic thereof.

[0048] In some further embodiments, the peptide or mimetic thereof is a peptide which comprises two or more copies of an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36 (eg a dimeric or trimeric form) and which may or may not include a linker sequence or linker group as described above. Particular examples of such peptides include KAIHPMR-KAIHPMR (SEQ ID NO: 39) and KAIHPMR-GGGS-KAIHPMR (SEQ ID NO: 40).

[0049] In yet some further embodiments, the peptide or mimetic thereof is a peptide which comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36 and an FF dipeptide motif provided at the N-terminal but more preferably, C-terminal, of the peptide.

[0050] In some embodiments, the peptide or mimetic thereof may be provided in an isolated form, while in some other embodiments, the peptide or mimetic thereof may be provided as bound upon a solid support or substrate such as those described above. In addition, where the peptide or mimetic thereof is to be used in a method of the first aspect that is conducted using a flotation separation process, the peptide or mimetic thereof may be provided in a suitable gas (preferably air).

[0051] In some embodiments of the second aspect, the peptide or mimetic thereof may also be selected on the basis of hydrophobicity (ie the peptide or mimetic thereof may be relatively hydrophobic) and / or modified so as to be relatively hydrophobic, as has been described above.

[0052] The peptide or mimetic thereof of the present disclosure may also be of use in the analysis of the mineral content of a material, especially an ore sample or ore preparation (ie in a mineral diagnosis method). Typical current approaches to ore analysis involve a variety of techniques including thermogravimetric analysis, X-ray fluorescence spectrometry (XRF) and scanning and / or transmission electron microscopy (SEM and TEM). These techniques are complex, laborious and cannot be readily performed "in the field", meaning that there can be substantial delays (eg days or weeks) before, for example, an ore sample may be analysed and the results returned. It is considered that a peptide or mimetic thereof according to the present disclosure may enable a relatively quick assay of a material and may require only relatively simple and / or portable equipment which may therefore be conducted "on site" or in a nearby laboratory.

[0053] Thus, in a third aspect, the present disclosure provides a method of assaying a material for the presence of at least one mineral, wherein the method comprises contacting the material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, determining peptide or mimetic thereof that is bound to the desired mineral, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.

[0054] The material to be assayed may be, for example, a sample of an ore, ore preparation (eg an ore that may have been, for example, crushed or otherwise comminuted, sieved to comprise particles of a desired size, concentrated using any of the standard methodologies known to those skilled in the art such as gravity concentration and froth flotation, and / or subjected to a leaching step), tailings, tailings preparations (eg tailings that may have been, for example, crushed or otherwise comminuted, sieved, concentrated, and / or subjected to a leaching step) and tailings dam water and / or settled materials from the bottom of a tailings dam. The material may be provided in a fluid form, preferably as a liquid. However, in some embodiments, the assay may use solid samples of the material (eg a thin wafer cross-section sample from a core sample) and the step of contacting the material with the mineralbinding peptide or mimetic thereof is conducted by contacting the peptide or mimetic thereof with thesurface of the solid sample. The binding of the peptide or mimetic thereof may then be determined in situ on the sample surface.

[0055] In some embodiments, the peptide or mimetic thereof is provided with a detectable label (eg a radioisotope, fluorophore, hapten or enzyme) as are well known to those skilled in the art, to enable the determination of peptide or mimetic thereof that is bound to the desired mineral by, for example, qualitative or quantitative detection methodologies. For example, in one particular embodiment, the peptide or mimetic thereof may be conjugated with a fluorescent dye such as cyanine5 (Cy5), 5- carboxytetramethylrhodamine (TAMRA) and Alexa fluor 647.

[0056] In some other embodiments, the binding of the peptide or mimetic thereof may be determined by using a reagent which binds to the peptide or mimetic thereof and, in so doing, generates a detectable signal. For example, fluorescamine reacts with primary amines to form highly fluorescent products, thereby making it useful as a Anorogenic agent for the detection of peptides (ie via their amine groups).

[0057] The peptide or mimetic thereof may be modified for use in the method of the third aspect by, for example, C-terminal amidation, modification to generate a peptide comprising more than one copy of the amino acid sequence (eg a dimeric or trimeric form) wherein the copies of the amino acid sequence is / are repeated in a head to tail arrangement or joined by a short linker sequence or linker group as described above, and / or the addition of an FF dipeptide motif which may enhance the binding affinity of the peptide for the mineral(s).

[0058] In some embodiments, the method of the third aspect may be conducted using multiple different peptides or mimetics thereof to enable the assaying of two or more minerals. Such a method may, for example, comprise contacting the material simultaneously with a first peptide or mimetic thereof which selectively binds to a first mineral (eg silver) and a second peptide or mimetic thereof which selectively binds to a second mineral (eg gold), wherein the first and second peptides or mimetics thereof are provided with different detectable labels so as to enable thereafter, the separate determination of binding of the respective peptide or mimetic thereof to the respective mineral to be assayed.

[0059] As will be appreciated by those skilled in the art, the conditions under which the material is contacted with the peptide or mimetic thereof may be adjusted so as to optimise the binding of the peptide or mimetic thereof with the desired mineral(s) contained in the material. However, typically, the conditions used will include: a temperature in the range of 4 to 40 °C, more preferably 10 to 30 °C, and most preferably 15 to 30 °C; and a pH in the range of 4 to 8, but more preferably 5 to 7. In some embodiments, the conditions under which the material is contacted with the peptide or mimetic thereofmay comprise a salt concentration in the range of 5 to 10 wt% (based upon the total weight of the fluid material).

[0060] It will be readily appreciated by those skilled in the art that the methods, peptides and peptide mimetics of the present disclosure are not restricted in their use to the particular application described. Neither is the methods, peptides and peptide mimetics restricted in their preferred embodiment(s) with regard to the particular elements and / or features described or depicted herein. Further, it will be readily appreciated that the methods, peptides and peptide mimetics are not limited to the embodiment(s) disclosed, but are capable of numerous rearrangements, modifications and substitutions without departing from the scope of the present disclosure. For example, the novel use of multimeric forms (eg dimeric and trimeric forms) of a mineral-binding peptide or mimetic thereof may be advantageous in methods of separating at least one desired mineral from a material or for assaying a material for the presence of at least one mineral (ie mineral diagnosis), and need not be restricted to peptides comprising a 7-mer amino acid sequence from those shown as SEQ ID NOs: 1 to 36 (or mimetics thereof). That is, it is to be understood that the present disclosure also extends to the use of multimeric forms, preferably dimeric or trimeric forms, of other mineral-binding peptides such as those that have been previously described (eg nickel- and cobalt-binding 9-mer peptides described in Matys S et al., J Environ Chem Eng 8:103606, 2020, gallium-binding 12-mer peptides described in Schonberger N et al., Biomimetics 4, 35; doi: 10.3390, 2019, molybdenum-binding peptides described in Centinel S et al., Scientific Reports 8:3374, 2018, and the lanthanum-binding peptides described in Lederer FL et al., Minerals Eng 132:245-250, 2019) or which may be identified by further screening of short random peptides (whether 7-mer peptides or of another length, such as 8-mers, 9-mers, 10- mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers and 20-mers) with a phage display-based method such as that disclosed herein.

[0061] Thus, in a further aspect, the present disclosure provides a method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide or mimetic thereof is provided in a multimeric form (eg comprising more than one "copy" of the amino acid sequence (which may be, for example, 5 to 25 or more, preferably, 7 to 15 amino acids in length), and wherein the copies of the amino acid sequence is / are repeated in a head to tail arrangement such that the C-terminal residue of a first copy of the amino acid sequence is adjacent the N-terminal residue of the second copy of the amino acid sequence, or wherein more than two copies of the amino acid sequence are contemplated, the C-terminal residue of the second copy of the amino acid sequence is adjacent the N-terminal of a third copy of the amino acid sequence, and so on, or wherein the copies of the amino acid sequence is / are joined by a short linker sequence or linker group).

[0062] In a yet further aspect, the present disclosure provides a method of assaying a material for the presence of at least one mineral, wherein the method comprises contacting the material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, determining peptide or mimetic thereof that is bound to the desired mineral, wherein the peptide or mimetic thereof is provided in a multimeric form (eg as described above in the preceding paragraph).

[0063] And in a yet still further aspect, the present disclosure provides a method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide or mimetic thereof is provided with an elastin-like polypeptide (ELPs) and wherein the said separating of the mineral(s) bound to the peptide or mimetic thereof comprises elevating the temperature of the fluid material to form separable aggregates of mineral(s) bound to the peptide or mimetic thereof.

[0064] In some embodiments, the method may further comprise recovering the peptide or mimetic thereof (as provided with an ELP) for re-use. The peptide or mimetic thereof may be a peptide of, for example, 5 to 25 or more, preferably, 7 to 15 amino acids in length (eg 7-mer peptides or of another length, such as 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers and 20-mers) or a mimetic thereof, which may be identified by, for example, further screening of short random peptides with a phage display-based method such as that disclosed herein. Further, the peptide or mimetic thereof may comprise a multimeric form (eg comprising more than one "copy" of the peptide or mimetic thereof) as described herein.

[0065] The said separating of the mineral(s) bound to the peptide or mimetic thereof preferably comprises elevating the temperature of the fluid material by about 2°C to more than about 25°C (eg about 30°C, 35°C or 50°C), and more preferably, elevating the temperature of the fluid material by about 5°C to more than about 25°C (eg about 20°C). The formed aggregates of mineral(s) bound to the peptide or mimetic thereof may be readily isolated / separated from the fluid material by any process that would be apparent to those skilled in the art (eg filtering or sieving).

[0066] The methods, peptides and peptide mimetics of the present disclosure is hereinafter further described with reference to the following non-limiting examples and accompanying figures.EXAMPLE(S)Example 1Methods and Materials

[0067] Phage displayThe M13 phage libraries displaying random 7-amino acid (7-mer) peptides (Ph.D.™-7 Phage Display Peptide Library Kit; New England Biolabs (Australia) Pty Ltd, Notting Hill, VIC, Australia) were used to conduct phage display as follows.

[0068] Briefly, an empty microcentrifuge tube and another microcentrifuge tube containing 2 mg silver powder were washed by TBST_0.1 (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5) twice, followed by blocking using blocking buffer (0.1 M NaHCOs, 5 mg / mL BSA, 0.02% NaNs, pH 8.6) on a rotary-vibrating mixer at 4 °C for 1 hour. Then, the empty microcentrifuge tube and another microcentrifuge tube containing silver powder were washed by TBST_0.1 four times. The phage library was first incubated with the empty microcentrifuge tube on a rotary-vibrating mixer for 20 min, then transferred to silver powder and allowed to incubate for another 20 min on a rotary-vibrating mixer. Afterward, the phage library was removed, and the silver powder was washed by TBST_0.1 10 times. The bound phages were eluted by elution buffer (0.2 M Glycine-HCl, 1 mg / mL BSA, pH 2.2), followed by immediate neutralisation by Tris-HCl (1 M, pH 9.1). The eluted phages were titered and amplified by adding to E. coli K12 ER2738 (New England Biolabs (Australia) Pty Ltd) culture at 37 °C for 4.5 h. Amplified phages were collected by centrifugation and precipitated by a precipitation buffer (20% PEG, 2.5 M NaCl) twice. The phage pellet was resuspended in TBS (50 mM Tris-HCl, 150 m NaCl, pH 7.5) as the new library followed by titering for the next round of screening. After three to five "biopannings", the amplified phages were diluted and grown on agar plates (1.5% agar, 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 50 ppm IPTG, 40 ppm X-Gal). Single plaques were picked and added to E. coli K12 ER2738 culture at 37 °C for 4.5-5 hours. Phages were purified by centrifugation and removal of the bacteria pellet. Thereafter, the phages were precipitated by a precipitation buffer (20% PEG, 2.5 M NaCl), and then resuspended by 100 ,uL iodide buffer (4 M Nal, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0), followed by the addition of 250 |1L ethanol and incubated for 15 min. The suspension was centrifuged at 20,000 g at 4 °C for 10 min and the supernatant discarded. The pellet was washed with 500 |1L of -20 °C 70% ethanol and centrifuged again, followed by the removal of the supernatant. The DNA pellet was dried under vacuum and redissolved in water. The isolated DNA was then sequenced after mixing with primers.

[0069] Isothermal titration calorimetry (ITC)ITC experiments were performed using a MicroCai PEAQ-ITC (Malvern Panalytical Ltd, Malvern, United Kingdom). In a standard measurement of a peptide Agl peptide named Agl (KAIHPMR; SEQ ID NO: 1) and AgNOs, Agl and AgNO? were dissolved in 25 mM HEPES buffer (pH 7) at 500 and 50 pM, respectively. Before loading samples, the syringe and the sample cell were rinsed with Agl and AgNOs samples, respectively. Agl was loaded in the syringe, and AgNO? was loaded inside the sample cell. Agl was injected into AgNO? via 19 injections. The first injection volume was 0.4 pL, and the volume of the rest injections was 2 pL. The injection time was 2 s, and the injection interval was 150 s. The temperature was set at 25 °C, and the stirring speed of the syringe was 750 rpm. The reference power was 10.0 pcal / s. Dissociation constant (KD), stoichiometry of binding (N), and thermodynamic parameters including enthalpy change (AH), entropy change (-TAS), and Gibbs free energy change (AG) were obtained according to a single -site binding model using the MicroCai PEAQ-ITC analysis software (Malvern). Data were averaged from three independent measurements.

[0070] Synthesis of Ag nanoparticles (NPs)The modified Tollens process was adapted to synthesise Ag NPs (Panacek A et al., Nat Nanotechnol 13:65-71, 2018). The final concentrations of all the reaction components were as follows: AgNO? 1 mM; NH3 H2O 5 mM; NaOH 2 mM, and D-maltose 10 mM. First, 0.5 mL of 10 mM AgNO? was added to 3 mL water, followed by the addition of 0.5 mL of 50 mM NH3 H2O and stirring (500 rpm) for 1 min. Afterward, the mixture was added with 0.5 mL of 20 mM NaOH and was stirred (500 rpm) for 1 min. Finally, 0.5 mL of 100 mM D-maltose was added to the mixture and stirred (500 rpm) for 5 min, followed by a static incubation of 60 min. After the incubation, the synthesised Ag NP suspension was dialysed against 25 mM HEPES (pH 7) using a molecular weight cut-off (MWCO) of 10 kDa dialysis tubing at 4 °C for 16 h. After dialysis, the concentration of Ag NPs was determined by inductively coupled plasma mass spectrometry (ICP-MS). The size of the Ag NPs was about 79 nm.

[0071] Synthesis of SiQ? NPsThe modified Stober method was adapted to synthesise SiCL NPs (Stober W et al., J. Colloid Interface Sci 26:62-69, 1968). The final concentrations of all the reaction components were as follows: EtOH 13.7 M; NH3 H2O 0.6 M; TEOS 0.26 M. First, 4 mL of EtOH was added to 0.514 mL water, followed by the addition of 0.196 mL of 15.32 M NH3 H2O and stirring (500 rpm) for 1 min. Afterward, the mixture was added with 0.29 mL of TEOS and stirred (500 rpm) for 60 min. Finally, the synthesised SiOz NP suspension was dialysed against 25 mM HEPES (pH 7) using a molecular weight cut-off (MWCO) of 10 kDa dialysis tubing at 4 °C for 16 h. After dialysis, the concentration of SiO NPs was determined by inductively coupled plasma mass spectrometry (ICP-MS). The size of the Si NPs was about 352 nm.

[0072] Dynamic light scattering (PLS)The hydrodynamic sizes and Zeta-potentials of all NPs were measured by DLS using a Zetasizer Nano ZS (Malvern Panalytical Ltd) at 25 °C with a scattering angle of 173°.

[0073] Ultra violet- visible (UV-Vis) spectroscopyThe absorbance of all NPs was measured by UV-Vis using a UV-2700 spectrophotometer (Shimadzu Scientific Instruments (Oceania) Pty. Ltd., Rydalmere, NSW, Australia) at 25°C.

[0074] Transmission electron microscopy (TEM)The morphologies of all NPs were observed by an FEI Tecnai G2 Spirit TEM (Thermo Fisher Scientific Inc., Waltham, MA, United States of America) operated at 100 kV. To prepare samples, 10 pL of NP suspension was placed on formvar-coated copper grids (ProSciTech Pty. Ltd., Kirwan, QLD, Australia) for 2 min, followed by removal using tissue and air-dried.

[0075] Selective precipitation of Ag NPs from Ag+SiCL NPsPurified Ag and SiO NPs were adjusted to concentrations of 75 and 214.29 mg / L in 25 mM HEPES (pH 7), respectively. Various peptides were dissolved in 25 mM HEPES (pH 7) to a final concentration of 10 mg / L. 2 mL of 75 mg / L Ag NPs were added with 0.7 mL of 214.29 mg / L SiO NPs and mixed well. Afterward, 0.3 mL of 10 mg / L peptide solution was added and mixed well. The mixture was maintained undisturbed and sampled at 16 and 24 h for ICP-MS measurement.

[0076] Inductively coupled plasma mass spectrometry (ICP-MS)The concentrations of Ag and Si were determined using an 8900 Triple Quadrupole ICP-MS (Agilent Technologies Australia, Mulgrave, VIC, Australia). Ag and SiO NPs were dissolved in 62.1% HNOs for 24 h, followed by dilution to different concentrations. The final samples were dissolved in 5% HNO3.Results and Discussion

[0077] A phage display method was used for screening mineral particles. Taking silver powder as an example, briefly, a phage library displaying 109different peptides was mixed with the silver to allow binding, then the weak-bound and unbound ones were washed away, followed by the elution of strong-bound ones. The eluted strong-bound phages were amplified in E. coli to form a new library of strong-bound phages, and then mixed with silver to do a second cycle (biopanning). After three to five biopanning cycles, the phages were purified and sequenced to obtain the amino acid sequences of the displayed peptides. The method is schematically depicted in Figure 1.

[0078] Four different amino acid sequences were obtained from the silver powder screening, with the highest frequency (of occurrence) peptide named Agl (KAIHPMR; SEQ ID NO: 1), and the three other peptides named Ag2 (NSMKHVH; SEQ ID NO: 2), Ag3 (HPITRHK; SEQ ID NO: 3), and Ag4 (RPTKMHR; SEQ ID NO: 4) (see Table 1). The four peptides showed diverse silver binding frequencies. Agl, with a 69% sequencing frequency, was then designed to mimic its phage display orientation with a free N-terminus and an amidated C-terminus for facilitating silver interaction. As shown in the following structure, the Agl peptide includes a sulphur atom in the methionine (M) and an imidazole ring in the histidine (H), both of which are believed to be favourable for silver coordination, while the amino groups in the lysine (K) and arginine (R) are also likely to contribute to binding.

[0079] Table 1 Peptides screened from silver powderMolecular Net chargeMineral Name Sequence SEQ ID No Frequency pH (I) weight at pH 7Agl KAIHPMR SEQ ID NO: 1 69% 851 11.5 3.1Ag2 NSMKHVH SEQ ID NO: 2 19% 850 10.0 2.2SilverAg3 HPITRHK SEQ ID NO: 3 6% 887 11.5 3.2Ag4 RPTKMHR SEQ ID NO: 4 6% 924 12.5 4.1

[0080] Apart from silver, other minerals were also screened using the phage display method. This enabled the identification of various mineral-binding peptides for gold (Au), quartz (Si), magnetite (Mag), and hematite (Hem) as summarised in Table 2 below. Interestingly, one peptide, namely HemlMag2 (WSLGYTG; SEQ ID NO: 21), was identified from the screening with both hematite andmagnetite. This peptide was also subsequently found to bind to the REEs, neodymium and dysprosium.

[0081] Table 2 Peptides screened from different mineralsMineral Name Sequence SEQ ID No Mol wtAul HSTAPQL SEQ ID NO: 5 752Au2 TGFPPYH SEQ ID NO: 6 817Au3 LYASRVP SEQ ID NO: 7 804Au4 GTGAYDA SEQ ID NO: 8 653Au5 KAQTLPA SEQ ID NO: 9 727Au6 FNNKHSS SEQ ID NO: 10 832Gold Au7 LVTVHYS SEQ ID NO: 11 817Au8 TAIRDEL SEQ ID NO: 12 816Au9 NHLDDAK SEQ ID NO: 13 811AulO LSLRPIP SEQ ID NO: 14 794Anil SYVGHSP SEQ ID NO: 15 745Aul2 LPTKPQL SEQ ID NO: 16 795Aul3 VAAREYS SEQ ID NO: 17 794Sil HFKLPKK SEQ ID NO: 18 896QuartzSi2 KPLKLPR SEQ ID NO: 19 850Magi GSLGYTG SEQ ID NO: 20 653MagnetiteHemlMag2 WSLGYTG SEQ ID NO: 21 782HemlMag2 WSLGYTG SEQ ID NO: 21 782Hem2 VAVPLHN SEQ ID NO: 22 748HematiteHem3 HSWRAPY SEQ ID NO: 23 915Hem4 FNISSSR SEQ ID NO: 24 809Hem5 NHAQFFK SEQ ID NO: 25 890Hem6 SHLIAER SEQ ID NO: 26 824Hem7 HFHPLES SEQ ID NO: 27 865Ndl WSLGYTG SEQ ID NO: 21 782Nd2 IYAKAPI SEQ ID NO: 28 775Rare earthNd3 LPPYELH SEQ ID NO: 29 868 neodymium (Nd)Nd4 HIGAAYE SEQ ID NO: 30 760Nd5 GSPYLFV SEQ ID NO: 31 782Rare earthDyl WSLGYTG SEQ ID NO: 21 782 dysprosium (Dy)Dy 2 HPSTWHK SEQ ID NO: 32 892Dy3 FPVVTRN SEQ ID NO: 33 832Dy4 QSFGPHP SEQ ID NO: 34 769Dy5 SYSLNPF SEQ ID NO: 35 827

[0082] Next, the silver-binding peptide Agl was chemically synthesised and its binding affinity to different silver materials verified. In particular, Agl was first investigated for its binding affinity to silver ions (Ag+) sourced from AgNO ? using ITC. The peptide (500 pM) in the syringe was injected into the AgNO? solution (50 pM) in the sample cell via 19 injections. A typical exothermic reaction was observed (see Figure 2a), demonstrating an enthalpy-driven binding affinity between Ag+and Agl. Control experiments were also conducted. The dilution effect of Ag+or Agl did not yield any significant thermic change (Figure 2b, c). Also, mixed NaNO? and Agl did not detect obvious binding activity (Figure 2d), which indicates that the binding between AgNO? and Agl resulted from Ag+-Agl interactions instead of NOs -Agl interactions.

[0083] Thermodynamics information was derived from the binding between Ag+and Agl ; with the disassociation constant being KD of 1.2*105, the association (binding) constant KA of 8.1*104, the binding stoichiometry N (Ag+per Agl) being 1.4, and an enthalpy change AH of -37.2 kJ / mol, entropy change -TAS of 9.2 kJ / mol, and a Gibbs free energy change AG of -28.0 kJ / mol (see Table 3).

[0084] Table 3 Thermodynamics of AgNCh + AglAH -TAS AGKD KA N (Ag+ / Agl)(kj / mol) (kj / mol) (kj / mol)1.2*1058.1*1041.4 -37.2 9.2 -28.0

[0085] In addition, an investigation was conducted to assess the impact of different parameters including temperature, pH and salt concentration on the binding between Ag+and the Agl peptide. It was found that with the increase of temperature from 4 to 37 °C, the KA significantly monotonically decreased from 2.6*105to 6.1*104, respectively (see Figure 3a), however, the binding site number N (Ag+ / Agl) did not show any obvious changes (Figure 3b). pH also had a great influence on binding. That is, shifting pH 5 to 7 yielded evident binding, whereas another pH unit drop eliminated the binding; therefore, the binding is highly pH-responsive (Figure 3c). Similarly, within the binding pH range (ie pH 5-7), the N (Ag+ / Agl) did not change much with varying pH (see Figure 3d).

[0086] The pH effect was also investigated using a series of different salt concentrations (ie 0, 1, 2, 5, 10 and 20 wt%) at pH 7 using NaNOs, which has been demonstrated to have no binding affinity to Agl (Figure 2d). Interestingly, both the KA and N were affected by salt non-monotonically (see Figure 2e, f) within the low salt concentration range (ie <5%), whereas the higher salt concentration range resulted in higher KA. This is probably because the stronger ionic strength of the solution screened partial charges of Ag+and cationic Agl, reducing their electrostatic repulsion since both are positively charged at pH 7. However, further increases in salt concentration rapidly reduced the binding constant from 7.4*105at 5% to 5.7*104at 20%. On the other hand, the N (Ag+ / Agl) showed an inversed varying trend compared to KA.

[0087] Based on the Agl peptide, peptide variants were designed to elucidate how different sequence modifications would affect the binding affinity. The peptide variants are shown in Figure 4. In a first example, and in order to better mimic the displayed peptide status on the phage, an Agl variant was designed (NH2-KAIHPMR-CONH2) without N-terminal modification but with C-terminal amidation to remove the C-terminal negative charge, as the displayed peptide on the phage is the start of a minor coat protein (pill) of Ml 3 phage and thus has a free cationic amine group at the N-terminus but a noncharged amide at C-terminus (Ph.D.™-7 Phage Display Peptide Library Kit: https: / / www.nebiolabs.com.au / products / e8100-phd-7-phage-display-peptide-library- kit#Product%20Information). Additionally, in phage display, the displayed peptides were connected to the phage coat protein by a tetrapeptide linker GGGS (SEQ ID NO: 37) which could enable the displayed heptapeptides higher flexibility. Therefore, Agl -GGGS (NH2-KAIHPMR-GGGS-CONH2; SEQ ID NO: 42) was designed to investigate the function of the linker. Phenylalanine (F) is an aminoacid with the side chain being a benzyl group, and which has a high propensity for self-assembly to different nanostructures via hydrophobic interactions and 71-71 stacking (Stankovic I et al., Int J Biol Macromol 156:949-959, 2020), so a further variant, designated Agl-2F (NH2-KAIHPMR-FF-CONH2; SEQ ID NO: 38) was designed to explore the F effect on the binding. In another approach to increase hydrophobicity, short hydrophobic peptide sequences (namely, AAIV (SEQ ID NO: 45) and VVLGAAIV (SEQ ID NO: 46)) were added to the Agl peptide (creating the variants named Agl- Pho4 and Agl-Pho8). It was also hypothesised that multiple copies could potentially increase the binding affinity of the peptides to Ag+(Zhang G et al., Bioconjugate Chem 14:86-92, 2003; and Dijkgraaf I et al., Eur J Nucl Med Mol Imaging 34:267-273, 2007), therefore dimeric peptide variants Agl-Agl (NH2-KAIHPMR- KAIHPMR-CONH2; SEQ ID NO: 39) and Agl-GGGS-Agl (NH2- KAIHPMR-GGGS-KAIHPMR-CONH2; SEQ ID NO: 40) were designed to examine whether the binding affinity can be improved. Besides, based on the simplest amino acid glycine (G) with only a hydrogen atom as the side chain, a control peptide 7G (NH2-GGGGGGG-CONH2; SEQ ID NO: 41) was produced to assess the specificity of Agl, along with another control peptide, comprising the linker GGGS (NH2-GGGS-CONH2; SEQ ID NO: 37), to evaluate the effect of the linker itself. The various Agl peptide variants are summarised in Table 4 below.

[0088] Table 4 Agl peptide variants and control peptidesN- C-Name Sequence SEQ ID No terminus terminusAgl NH2- KAIHPMR SEQ ID NO: 1 -CONTEAgl-2F NH2- KAIHPMR- FF SEQ ID NO: 38 -CONTEAgl-Agl NEE- KAIHPMR- KAIHPMR SEQ ID NO: 39 -CONTEAgl- KAIHPMR-GGGS-NEE- SEQ ID NO: 40 -CONTEGGGS-Agl KAIHPMR7G NIE- GGGGGGG SEQ ID NO: 41 -CONTEGGGS CI 1, CONI 1- GGGS SEQ ID NO: 37 -CONTEAgl-GGGS NIE- KAIHPMR-GGGS SEQ ID NO: 42 -CONTEKAIHPMR-GGGS-Agl-Pho4 NIE- SEQ ID NO: 43 -CONTEAAIVKAIHPMR-GGGS-Agl-Pho8 NH2- SEQ ID NO: 44 -CONH2WLGAAIV

[0089] ITC was used to examine the binding affinities of the Agl variant and control peptides to Ag+(see Figure 5). In general, all of the Agl peptide variants demonstrated clear-exothermic binding isotherms (Figure 5b-f). On the contrary, the two control peptides 7G and GGGS (SEQ ID NO: 37) did not show any binding affinity to Ag+(Figure 5g, h). Specifically, the different variants exhibited different thermodynamics. All Agl peptide variants revealed greater KA, N (Ag+ / peptide), AH, -TAS, and AG than those of Agl (Figure 5i-m). The linker-containing Agl variants (ie Agl-GGGS and Agl- GGGS-Agl) exhibited greater thermodynamics parameters than their non-linker counterparts (Agl and Agl -Agl), respectively. This could be due to the higher flexibility endowed by the linker when the peptides and Ag+bind and assemble, though the linker GGGS (SEQ ID NO: 37) itself did not show any binding affinity to Ag+(Figure 5h). The Agl-2F peptide variant did not lose the Ag+-binding affinity, and its binding affinity was even higher than that of Agl. The hydrophobic modifications in Agl-Pho4 and Agl-Pho8 increased binding affinity data not shown), with Agl-Pho8 exhibiting the lowest Kd of 8.3 pM. As hypothesised, the dimeric peptides (ie Agl-Agl and Agl-GGGS-Agl) showed stronger binding affinities compared to their monomeric counterparts (Agl and Agl-GGGS), respectively. Stoichiometry data indicated that Agl-Pho8 and the dimeric forms of Agl (eg Agl-Agl) bind multiple Ag+per molecule. Similar to the greater binding affinity shown by Agl-2F over Agl, the peptide dimers have a closer distance between the Agl peptide components, thus allowing more interactions between the Agl peptides sequences and Ag+.

[0090] After investigating the binding affinity between Ag+and the various peptides, the peptides were then examined to determine whether they can also bind to other silver materials such as Ag nanoparticles (NPs). Agl peptide variants were mixed with Ag NPs. However, a rapid colour change from orange to grey of the Ag NP suspension was observed, suggesting the aggregation of Ag NPs. Therefore, the size change of the Ag NPs was immediately measured upon the addition of peptides using DLS (see Figure 6a). The Agl peptide and all Agl peptide variants caused a significant size increase from 84 to 152, 153, 177, 261, and 259 nm for Agl, Agl-GGGS, Agl-2F, Agl-Agl, and Agl-GGGS-Agl, respectively, indicating a strong binding affinity to Ag NPs of these peptides. In contrast, the two control peptides, 7G and GGGS (SEQ ID NO: 37), did not have any impact on Ag NP aggregation. Interestingly, the degree of the size increase induced by the different peptide variants showed similar trends with their binding affinities (Figure 5). UV-Vis spectroscopy revealed a substantial red-shift of the absorbance peak from 400-500 to 700-800 nm for the Agl peptide variants (see Figure 6b), indicating that the aggregation of Ag NPs was due to the size-dependence surface plasmon resonances of Ag NPs (Heath J. Phys Rev B 40:9982, 1989), whereas the 7G and GGGS(SEQ ID NO: 37) peptides did not shift the absorbance at all. In addition, morphology change of the Ag NPs aggregated by different peptides using TEM was observed (Figure 6c). The original Ag NPs and those added with 7G and GGGS (SEQ ID NO: 37) exhibited spherical, uniform and monodispersed NPs, whereas the Agl peptide variants aggregated the Ag NPs into NP clusters. The sizes of the clusters agreed well with the DLS results (Figure 6a).

[0091] After confirming that the Agl peptide variants also have binding affinity to not only Ag+but also Ag NPs, the specificity of the peptides was then investigated. Here silica (SiOz) NPs were used as a model representing other NPs. The sizes of the Ag NPs and SiO NPs used in the experiments were 83 and 362 nm, respectively (see Figure 7a). Both had very low polydispersity index (PDI) values <0.1 (Figure 7b), suggesting that they had a narrow size distribution and were monodispersed. In addition, the surface charges of the Ag NPs and SiO NPs were both negative as evidenced by the Zeta-potentials of -43 and -54 mV (Figure 7c). Next, the Ag NPs and SiO NPs were mixed at the same mass concentration to examine whether the silver-binding peptides can selectively precipitate Ag NPs out. The final concentrations of Ag NPs, SiO NPs, and peptides were controlled at 50, 50, and 1 mg / E, respectively. After 16 h, the NP mixture without peptides, NP mixture + 7G, and NP mixture + GGGS (SEQ ID NO: 37) revealed a stable-orange NP suspension (Figure 7d). In contrast, the monomeric peptides (Agl, Agl -GGGS, and Agl-2F) already precipitated the majority of Ag NPs which formed black precipitates on the bottom. But the dimeric peptides (Agl-Agl and Agl-GGGS- Agl) seemed to only aggregate Ag NPs instead of precipitating them as the suspensions looked brown and opaque. Quantitative analysis of the Ag and Si distributions in suspension and precipitates using ICP-MS (Figure 7e, f) agreed well with their appearances. The separation factor (Ag / Si) was calculated as follows: Ag mass in precipitate (Pm^)Ag o distribution ratio ( vDAn) = - Ag mass i:n suspensi:on (Sm^) (1) i - -i z x SiO2mass in precipitate (Pms>)SiO2distribution ratio (Dsi) = SiO2mass in suspensi —on 7 ( —Sm / ) (2)

[0092] The Agl-2F, Agl, and Agl-GGGS peptides resulted in effective separations between Ag NPs and SiOz NPs, with separation factors of 13, 109, and 1416, respectively (Figure 7g). Further incubation to 24 h led to considerable precipitation of Ag NPs by Agl all Agl peptide variants (Figure 7h), while Ag NPs with 7G and GGGS (SEQ ID NO: 37) only were stable. ICP-MS confirmed the effective precipitation of Ag NPs by all Agl peptide variants (Figure 7i), with more than 87% Agprecipitation efficacy. Indeed, the precipitates induced by Agl variants contained only a small fraction of silica (<5.9%) with the majority comprising silver (>94.4%); achieving the highest silver purity of 98.3% using Agl-Pho8 in the precipitate. The separation factors of Agl variants, defined by the ratio of DAg / Dsi, Where DAg is mAg precipitated / m;\g suspended and Dsi is msi precipitated / Ulsi suspended, aTC within the range from 415 to 1,562, with the two highest values observed for Agl-Pho8 (1562) and Agl-GGGS- Agl (1123). The high separation factors demonstrate the peptides' superior selectivity in precipitating Ag NPs, highlighting their potential applications in the selective separation of mineral particles.

[0093] However, interestingly, the dimeric peptide variants, Agl-Agl and Agl-GGGS-Agl, were able to precipitate substantial quantities of SiO NPs (39 and 40%, respectively) after 24 h (Figure 7j), and the Agl and Agl-GGGS peptides demonstrated very high separation factors of 1317 and 707, respectively (Figure 7k).

[0094] The surprisingly significant SiOz-aggrcgating ability of the dimeric peptide variants (Agl-Agl and Agl-GGGS-Agl) led to the consideration of whether this was due to the dimers possibly having a particular sequence of amino acids that can target and aggregate SiO NPs. Therefore, the size change of three systems was monitored over 24 h: 1) Ag NPs only; 2) SiO NPs only; 3) a mixture of Ag NPs and SiOz NPs. The results are shown in Figure 8. It was found that all three systems without peptide and without the addition of 7G and GGGS, did not show any obvious change of either size or PDI over 24 h (Figure 8a, i, j), suggesting that there are no interactions between Ag NPs or SiO NPs and 7G or GGGS (SEQ ID NO: 37). For all other Agl peptide variants, similar trends were observed (Figure 8b- h). Ag NPs only and Ag NPs+SiO NPs systems exhibited significant size (solid lines) and PDI (dashed lines) increase over time, indicating that the aggregation of Ag NPs caused by the binding affinity between Agl peptide variants and Ag NPs. On the contrary, SiO NPs only were very stable over 24 h without obvious changes of size or PDI, suggesting that there was no binding affinity between Agl peptide variants and SiO NPs. Therefore, the capability for precipitating SiO NPs from Ag NPs+SiOz NPs of Agl-Agl and Agl-GGGS-Agl would appear not to be because the dimeric peptide design endows the SiO2-binding property. Instead, it is possibly caused by the different aggregating structures formed by Ag NPs, SiO2 NPs, and peptides, given that the precipitating periods were quite different for the monomeric and dimeric peptides (Figure 7d, h), indicating the formation of "looser" Ag NP aggregates induced by the dimers (Agl-Agl and Agl-GGGS-Agl) which can trap SiO2 NPs thus precipitating together with Ag NPs.Conclusion

[0095] A phage display-based method was successfully used to screen for short peptides able to bind to a target desired mineral(s). These peptides were then able to separate the targeted mineral (in this example, silver) from a model waste material, namely silica particles (nb. silica is a common waste material of, for example, the mining industry), with an exceptionally high separation factor (>1400), thereby showing the significant potential of this approach to the separation or recovery of desired, and potentially valuable, mineral(s) from various materials such as, for example, ore, e-waste and industrial wastewater. The binding of the target desired mineral(s) may be "tunable" by controlling various environmental conditions such as temperature, pH and ionic strength to ensure functionality across a spectrum of conditions.Example 2Methods and Materials

[0096] Fluorescamine and mineral particles including neodymium (III) oxide (NdzOs), dysprosium (III) oxide (DyzOs), kaolinite (AFSizOdOHF,). montmorillonite ((Na,Ca)o.33(Al,Mg)2(Si40w)(OH)2-nH20), quartz (SiCF), and gold (Au) were purchased from a commercial supplier. Fluorescamine reacts with primary amines to form highly fluorescent products, thereby making it useful as a Anorogenic agent for the detection of peptides (ie via their amine groups). The WSLGYTG (SEQ ID NO: 21) peptide (Ndl) was selected for use in this example as the diagnostic peptide.

[0097] Method for binding of diagnostic peptide to rare earth mineralsMaterials such as neodymium (III) oxide and kaolinite were dispersed in 100 pL HEPES buffer (25mM, pH 7.5) to produce suspensions containing 10 mg of the respective mineral. The diagnostic peptide was prepared in the same buffer at a concentration of 100 pM, while Auorescamine was dissolved in DMSO at 20 mM. Tween-20 solutions at 0.5% and 10% concentrations prepared using HEPES buffer (25 mM, pH 7.5). The 10% Tween-20 solution was added to the mineral sample to block non-specific binding, with the blocking process repeated twice. After the blocking step, centrifugation and washing with 0.5% Tween-20 solution were carried out three times. All of the washing and resuspension procedures were carried out in the presence of 0.5% Tween-20 solution. Then, the mineral suspensions were mixed with 100 pL of the diagnostic peptide solution and vortexed for 1 min. After centrifugation at 10,000 g for 1 min (to remove the supernatant), the powder was washed three times with 1 mL HEPES with 0.5% Tween-20. Thereafter, the pellet was resuspended in 180 pL buffer, mixed with 20 pL Auorescamine, and then vortexed for 1 min to allow for reaction with primary amine groups of the peptide. Further washing steps were then conducted,and the minerals subsequently resuspended and analysed using ZOE™ Fluorescent Cell Imager (BioRad Laboratories, Hercules, CA, United States of America) (Green Channel: Excitation: 480 / 17 nm; Emission: 517 / 23 nm). The final products were also examined using the fluorescent cell imager.

[0098] Testing of the method with different ratios of rare earth minerals and non-binding minerals Minerals were combined in different weight percentages: 10 wt% neodymium (III) oxide with 90 wt% kaolinite; 50 wt% neodymium (III) oxide with 50 wt% kaolinite; and 90 wt% neodymium (III) oxide with 10 wt% kaolinite. Peptide binding, washing and imaging procedures were performed substantially as described in the preceding paragraph.

[0099] Testing of the method with other rare earth mineralsThe method was also tested with other mineral examples, namely dysprosium (III) oxide, montmorillonite, quartz and gold. The preparation of these minerals, the peptide binding, washing and imaging procedures with fluorescamine were performed as described above.Results and Discussion

[0100] A typical REE oxide, neodymium (III) oxide (NdzOs), and a typical gangue mineral, kaolinite, were selected for testing for the potential of the mineral-binding peptides to be used diagnostically in assessing the mineral content of a material (eg in an ore sample). In particular, particles of the minerals were incubated with the mineral-binding peptide, unbound peptide washed away, and fluorescamine added to detect primary amine groups at the N-terminus of peptides.

[0101] To minimise any non-specific binding of the mineral-binding peptide to non-target minerals and materials such as kaolinite, a surfactant (Tween-20) was used to block the mineral surface before the peptide binding step. It was found that neodymium (III) oxide exhibited strong fluorescence, but kaolinite showed no fluorescence. This indicates that Tween-20 can block non-specific binding sites on the mineral surface (in this case, kaolite), thereby minimising the non-specific binding activities of the neodymium-binding peptide to mineral particles and enabling the contrast between target and nontarget minerals to be enhanced.

[0102] Using the diagnostic method, testing was also conducted to examine the binding affinity of the mineral-binding peptide to other mineral types, including dysprosium (III) oxide, other common gangue particles such as montmorillonite and quartz, and another metal particle, gold. As the Ndl peptide also binds to dysprosium (see Dyl in Table 1), it was found that the mineral-binding peptide bound dysprosium (III) oxide, although not as pronounced as that to neodymium (III) oxide. However, the mineral-binding peptide did not demonstrate binding affinity to any of the non-REE particlestested, namely kaolinite, montmorillonite, quartz, and gold, thus demonstrating high specificity for binding to REE particles.

[0103] In further testing, neodymium (III) oxide and kaolinite particles were mixed together at various ratios; in particular, 10 wt% NdzO? with 90 wt% kaolinite, 50 wt% NdjOs with 50 wt% kaolinite, and 90 wt% NdzO? with 10 wt% kaolinite. The mixtures were first blocked with Tween-20, followed by washing and incubation with the Ndl peptide, then washing once more before finally reacting with fluorescamine to create highly fluorescent signals. It was clear that only a small portion of particles showed fluorescence in the 10 wt% NdzO? plus 90 wt% kaolinite sample. In particular, only the black particles (NdzOs) were marked with fluorescence instead of the relatively transparent particles (kaolinite), indicating the specific binding affinity of the mineral-binding peptide to the particles of neodymium (III) oxide. With the proportional increase of NdzO ? to 50wt% and 90 wt%, the fluorescence signals intensified, reflecting the specific binding activity of the peptide to the NdzO? particles. It was also noted that the neodymium (III) oxide particles demonstrated significant aggregation in the absence of Tween-20, whereas the kaolinite appeared to remain well dispersed, with or without Tween-20. This confirms that Ndl peptide selectively binds to and aggregates NdzO? particles over the kaolinite particles, in a behaviour similar to that observed in Example 1 with the Agl peptide with silver and silica NPs.Conclusion

[0104] A method has been identified that enables the use of mineral-binding peptides for diagnostically assessing a material such as an ore sample for a target mineral such as an REE. The method particularly looked at the potential of the Ndl peptide which binds to both of the assayed REEs, that is neodymium (III) oxide and dysprosium (III) oxide. However, the method could be worked with an alternative peptide(s) to enable the separate diagnosis of a mineral sample for these REEs. The results achieved showed that a high level of specificity for the target mineral could be achieved with the mineral-binding peptides, thereby indicating their significant potential as precise and rapid diagnostic tools for materials such as mineral samples.Example 3Methods and Materials

[0105] Design and synthesis of Agl peptide variant, AMI -Agl A dual-functional Agl peptide variant, named AMI -Agl (Table 5), was designed and synthesised so as to enable an emulsion-based approach to particle separation. This peptide combines the silver- binding peptide Agl (KAIHPMR; SEQ ID NO: 1) with a peptide surfactant known as AMI describedin Dexter AF et al., Nat Mater 5(6):502-506 (2006). The AMI peptide exhibits surface-active properties and can facilitate the formation of oil-in-water emulsions (Wibowo D et al. , J Phys Chem C 121(27): 14658-14667 (2017)).

[0106] Table 5 Agl peptide variant, AMl-AglN- C-Name Sequence SEQ ID No terminus terminusMKQLADS-AMl-Agl CHaCONH- LHQLARQ- SEQ ID NO: 47 -CONH2VSRLEHA-KAIHPMR

[0107] Synthesis and characterisation of nanoemulsionsNanoemulsions were prepared using two distinct surfactants: a peptide surfactant, AMl-Agl (Ac- MKQLADS-LHQLARQ-VSRLEHA-KAIHPMR-CONH2; SEQ ID NO: 47), and a conventional surfactant, Tween 20. The peptide surfactant-based nanoemulsions were prepared by dissolving AMl- Agl in a 25 mM HEPES buffer at pH 7.0 to achieve a final concentration of 50 pM. ZnC12 was added to the same solution to reach a final concentration of 50 pM. Then, 980 pL of this peptide solution was mixed with 20 pL of the oil phase, 1 -octadecene. The mixture was then sonicated using a Branson Digital Sonifier SFX 550 (Emerson Automation Solutions, Bayswater, VIC, Australia) set to 10% amplitude. The process consisted of five bursts of 30 seconds each, with a 30-second ice bath cooling period in between each burst to prevent overheating. Similarly, Tween 20-based nanoemulsions were produced using a 50 pM Tween 20 solution. The synthesis followed the same oil-to-water ratio and sonication parameters as the peptide-based emulsions but without the addition of ZnCL. The size distribution of nanoemulsions was then characterised using DLS.

[0108] Nanoemulsion coalescence and NP separationAg and SiC NPs were first mixed at a concentration of 500 pg / mL each. Subsequently, 500 pL of the nanoemulsion was mixed with 1 mL NP mixture by stirring (200 rpm) for one minute. Following the mixing, the combined mixture was further mixed with 1.5 mL 2xPBS. This addition was observed to result in immediate coalescence of the peptide-stabilised nanoemulsion within seconds. In contrast, the Tween 20-based nanoemulsion remained stable without any emulsion coalescence or phase separation. Both emulsions were then allowed to settle for one hour. The upper layer of the peptide-stabilised emulsion was carefully removed. 1 mL sample was taken from the centre of the suspension for subsequent element analysis using ICP-MS.Results and Discussion

[0109] Rapid emulsion-based separation using a dual-functional AMl-Agl peptideTo further enhance the speed of particle separation, an emulsion-based approach was investigated using the dual-functional peptide, AMl-Agl. It was found that the fusion of AMI with Agl does not compromise its silver-binding capability (Figure 9a), while the integration of the AMI module leads to a reduced binding constant but enhanced binding sites (Figure 9b, c), as indicated by the changes in Kd and N. The AMl-Agl was also found to exhibit less negative AG and AH, but more favourable entropy changes.

[0110] The application of AMl-Agl in separating silver and silica NPs was demonstrated through an emulsion-based separation process. First, a stable emulsion with an average diameter of 292 nm was created using the AMl-Agl, due to its excellent surface activity. Then mixing this emulsion with silver and silica NP suspensions facilitated the specific binding of silver NPs to the emulsion surface. Upon introducing phosphate -buffered saline (PBS), the emulsion destabilised due to the stimuli -responsive property of AMI, which responds to changes in ionic strength (Li Y et al., Adv Fund Mater 33(7):2210387 (2023)). This destabilisation triggered a rapid creaming of the oil phase with Ag NPs bounded and rose to the top, separating effectively from the suspended SiO2 NPs. Then, the emulsions underwent a rapid phase separation upon PBS addition within seconds. In contrast, Tween 20 formed a stable emulsion that remained well dispersed after mixing with silver / silica suspensions.

[0111] The emulsion-based separation occurs much quicker, typically within a few seconds, compared to the aggregation-based method (described in Example 1), which can take up to 24 hours. The distribution of Ag and SiO2 NPs post-emulsion destabilisation was quantitatively analysed (Figure 9d, e). 96.0% of the Ag NPs floated to the top, whereas the majority (81.5%) of SiO2 NPs remained in suspension, achieving a separation factor of 106 (Figure 9f).Conclusion

[0112] The study described in this example highlights the great potential of dual-functional peptides or mimetics thereof for rapid, efficient and selective particle separation. The salt effects described above in Example 1 provides valuable design guidelines for applying the dualfunctional peptide, AMl-Agl. Optimal salt concentrations (<5 wt%) can enhance the binding affinity between the Agl peptide and silver, ensuring the successful flotation of Ag NPs with the oil phase upon emulsion destabilisation.Example 4Methods and Materials

[0113] Production of ELPsA polynucleotide molecule encoding an elastin-like protein (ELP) was obtained (Addgene, Watertown, MA, United States of America), while polynucleotide molecules encoding the Ag-binding peptides and the (GS)10 linker were purchased from Integrated DNA Technologies Australia Pty Ltd (Mount Waverley, VIC, Australia). A utility vector was initially created by fusing the linker at the C- terminus of the ELP protein along with multiple cloning sites for the insertion of other proteins or peptides using the Gateway assembly technique. Subsequently, oligonucleotides encoding the Ag peptides, complete with a stop codon, were cloned using the BamUI and Hindlll restriction enzymes. ELP proteins fused with either Agl or an Agl-Agl dimer were expressed in Escherichia coli (BL21) cells carrying the plasmid-borne genes. The expression was initiated following overnight induction with IPTG. Cultures for protein expression were started by inoculating 1 mL of the resuspended starter culture into each of four 800 mL flasks, which were then incubated at 37 °C with agitation at 200 rpm. Protein expression was induced at an GD600 of 0.6-0.7 by the addition of 0.5 mM IPTG to the culture medium. Following overnight incubation at 28°C, cells were harvested via centrifugation (5422 g, 10 min, 4°C) and subsequently resuspended in PBS buffer. Cell disruption was achieved through sonication. After sonication, the cell lysates were clarified by centrifugation (29097 g, 40 min, 4 °C), then NaCl was added at a final concentration of 2 M at 40 °C to precipitate the protein. The resultant white precipitate was then collected by centrifugation (29,097 g, 10 min, 40 °C), and the supernatant was discarded. The precipitate was redissolved in chilled PBS buffer and subjected to repeated cycles (at least three) of precipitation and dissolution — precipitation at 40 °C followed by dissolution at 4 °C. In the final step, the purified proteins were dissolved in 25 mM HEPES buffer, pH 7.0, containing 150 mM NaCl. The quality of the proteins was assessed using analytical size-exclusion chromatography.

[0114] Determination of phase lower critical solution temperature (LCST) of ELPsThe concentration of ELPs was adjusted to 500 pM for the assessment of their LCST. Absorbance at 350 nm was measured to monitor the phase transition using a plate reader (Infinite® 200 PRO, Tecan Group Ltd., Mannedorf, Switzerland) capable of temperature control. The temperature range for the measurements was set from 25 to 40 °C, with incremental increases of 1°C each to capture the precise temperature at which the ELPs undergo a phase transition, as indicated by a significant change in absorbance.

[0115] Separation of Ag and SiO? NPs using ELPsAg and SiO NPs were first mixed at a final concentration of 500 pg / mL each. Two ELPs, ELP-Agl (H6-SSGGVG-(VPGVG)59-VGVPWP-(GS)7-KAIHPMR; SEQ ID NO: 48) and ELP-Agl-GGS-Agl(H6-SSGGVG-(VPGVG)59-VGVPWP-(GS)7-KAIHPMR-GGS-KAIHPMR; SEQ ID NO: 49), were used to selectively separate the NP mixture (nb. The He moiety was included to allow for efficient purification as is well known to those skilled in the art (eg using metal affinity chromatography with a nickel column); the adjacent SSGGVG (SEQ ID NO: 51) hexapeptide provides flexibility to the He making it available, for example, for binding to a solid support (eg resin) during purification; the (GS)? moiety provides a linker / spacer between the ELP and the peptide or mimetic thereof, preventing steric hinderance and thereby allowing each to function independently; and the VGVPWP (SEQ ID NO: 52) hexapeptide enables characterisation of protein concentration, as it contains tryptophan (Trp) which has a specific absorbance at 280 nm, distinct from the general absorbance of peptide bonds (c / at 190- 230 nm). ELP-Agl was used at final concentrations of 0.2 and 1 pM, while ELP-Agl-GGS-Agl was used at final concentrations of 0. 1 and 0.5 pM. The control sample was set as the NP mixture without ELPs. The experiments were conducted under two temperature conditions: room temperature (22 °C) which is below the LCST of ELPs, and 40°C which is above the LCST. This setup was chosen to observe the efficacy of ELPs in nanoparticle separation under different thermal conditions. Photographs of the nanoparticle suspension were taken at various time points: immediately after mixing (time zero) and after 1, 2, 4, and 24 hours, to visually monitor the separation process. Samples were collected from the centre of the cuvette at these time points. Each sample of 1 mL was subsequently analysed using ICP-MS to quantify the concentrations of Ag and Si elements.

[0116] Recycling of ELPsThe ELP was recycled after it was used to separate Ag NP from the NP mixture at 40°C. The mixture was first cooled while shaking to transition unbound ELPs back to a soluble state as the temperature decreased below the ELP LCST. Then an acidic environment was introduced by adding a 0.1 M acetic acid buffer (pH 3) to disrupt the Ag-ELP interaction, causing the ELPs to desorb from the nanoparticles. Afterward, the mixture was centrifuged (20,000 g, 15 min, 4°C) to separate the soluble ELPs from the silver pellet. The pH of the supernatant containing ELPs was then adjusted back to 7 using 0.2 M NaOH. Then, the neutralised ELP solution was reheated to 40°C to trigger a hydrophobic transition, causing the ELPs to precipitate. This precipitated ELP was then collected by centrifugation (20,000 g, 15 min, 40 °C), and the ELP pellet redissolved using cold HEPES buffer for re-use. After recycling, the concentration of the recovered ELPs was measured using the Pierce™ BCA Protein Assay Kits. The recovery rate was calculated by comparing the quantity of ELPs before and after the recycling process. The functionality of the recovered ELP-Agl and ELP-Agl-GGS-Agl was examined by adjusting their concentrations to 1 and 0.5 pM, respectively. Freshly prepared ELPs with the same concentrations were used as control. Both the recovered and fresh ELPs were then used to induce precipitation with Ag NPs at 40 °C for 4 hours. Photographs and samples were then taken and analysed via ICP-MS to compare the efficiency of nanoparticle precipitation achieved by both fresh and recovered ELPs.Results and Discussion

[0117] Elastin-like polypeptides (ELPs) were investigated for the purpose of potentially enabling a cost-effective and recyclable separation of mineral particles. ELPs exhibit reversible solubility in response to temperature, a characteristic derived from the pentapeptide sequence VPGX'G (SEQ ID NO: 50) in tropoelastin. Two ELPs, ELP-Agl and ELP-Agl-GGS-Agl were designed incorporating such a pentapeptide and produced in E. coli using an inexpensive production method (Meyer DE and A Chilkoti, Nat Biotechnol 17(11):1112-1115 (1999); and Hussain Z et al., Adv Funct Mater 32(13): 2109158 (2022).

[0118] The temperature -responsive phase transition of ELPs is essential for the aggregation and separation of Ag NPs. That is, as temperature rises to the lower critical solution temperature (LCST) of 32.1 °C, ELP-Agl exhibits a significant increase in absorbance at 350 nm, indicative of its transition from soluble (hydrophilic) to insoluble (hydrophobic). It was hypothesised that the design of ELP- Agl-GGS-Agl would achieve a synergistic effect in separation by combining both the hydrophobic module and dimeric module. Although higher temperatures reduce binding affinity, Agl could retain silver-binding capabilities up to 45°C, enabling separation while heated. This underscores the role of ELP-Agl in efficient and temperature -responsive nanoparticle separation.

[0119] The performance of ELP-Agl and ELP-Agl-GGS-Agl in nanoparticle separation was assessed at varying concentrations and temperatures. While the blank controls showed stable particle suspension, the addition of ELPs induced Ag NP aggregation, especially at 40°C above LCST, where ELPs shifted to a hydrophobic state favourable for nanoparticle precipitation. Notably, ELP-Agl- GGS-Agl, with its dimeric structure, showed enhanced effectiveness, despite a lower ELP concentration compared to the ELP-Agl. This suggests that the dimerisation of the Agl peptide significantly enhances the separation efficacy.

[0120] The distribution of Ag and Si in the suspension solution and precipitates after treatment was also analysed (Figure 10 a, b). Notably, at a concentration of 0.5 pM ELP-Agl-GGS-Agl and a temperature of 40°C, 93.5% Ag NPs were aggregated and separated from the suspension. This high precipitation rate demonstrated the synergistic effect of temperature -responsive ELP behaviour and the enhanced binding properties of the dimeric Agl molecule. SiO NPs largely remained in suspension under all conditions, further highlighting the specificity of these Ag-binding ELPs. A maximum separation factor of 363 was achieved using ELP-Agl-GGS-Agl at 40°C and a 0.5 pM concentration within 4 h, which surpasses the efficiency of the Agl peptide (separation factor of 122) in double the time at 8 h (Figure 10c). This demonstrates the potential of ELP to accelerate the separation process. Further, a much higher separation factor of 23,004 was achieved after 24 h incubation due to the nearcomplete (99.9%) precipitation of Ag NPs by 0.5 pM ELP-Agl-GGS-Agl at 40 °C (Figure lOd).Their ability to achieve extremely high separation factors at relatively low concentrations, coupled with the flexibility to customise the separation process by adjusting temperature and peptide concentration, provides a versatile toolkit for particle separation.

[0121] In addition to their cost-effective production, temperature responsiveness, and high separation capability, ELPs also offer the advantage of recoverability owing to their reversible solubility transition. As Agl loses its silver binding affinity at pH below 4, the ELPs bound to precipitated Ag NPs can be effectively released by adjusting the pH to 3. Once the ELPs were released and neutralised, they can be effectively precipitated out upon heating to 40 °C, thus allowing for their recovery (Figure 11). High recovery rates of 97.4% and 92.1% were achieved for ELP-Agl and ELP- Agl-GGS-Agl, respectively (Figure 12a), and the recycled ELPs maintained their ability to precipitate Ag NPs effectively. The performance of fresh and recycled ELP peptides in precipitating Ag NPs within 4 h was further quantified (Figure 12b). The comparison demonstrates that the recycled ELPs can achieve Ag NP precipitation comparable to that of the freshly synthesised ELPs. This highlights not only the robustness of the ELPs through the recycling process but also their potential for multiple cycles of particle separation, offering the prospect of substantial cost reduction and waste reduction in industrial applications.

[0122] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0123] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[0124] It will be readily appreciated by those skilled in the art that the methods, peptides and peptide mimetics etc of the present disclosure are not restricted in their use to the particular application described. Neither are they restricted in their preferred embodiment(s) with regard to the particular elements and / or features described or depicted herein. Further, it will be readily appreciated that the methods, peptides and peptide mimetics etc are not limited to the embodiment(s) disclosed, but are capable of numerous rearrangements, modifications and substitutions without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.

2. The method of claim 1, wherein the peptide or mimetic thereof is provided on the surface of a solid support or substrate.

3. The method of claim 2, wherein the peptide or mimetic thereof is provided on the surface of suitable beads provided in a separation column.

4. The method of claim 1 , wherein the peptide or mimetic thereof is selected on the basis of hydrophobicity and / or modified so as to be relatively hydrophobic, and the fluid material is contacted with the peptide or mimetic thereof in a flotation separation process, the arrangement being such that the hydrophobic peptide or mimetic thereof assemble at the interface of gaseous bubbles provided for the flotation separation and a water solvent or carrier of the fluid material.

5. The method of claim 4, wherein the peptide or mimetic thereof further comprises a hydrophobic moiety or surfactant peptide.

6. The method of claim 5, wherein the hydrophobic moiety is an FF dipeptide moiety.

7. The method of claim 5, wherein the surfactant moiety is MKQLADS-LHQLARQ-VSRLEHA (SEQ ID NO: 53).

8. The method of any one of claims 1 to 7, wherein the peptide or mimetic thereof further comprises a temperature-sensitive peptide, oligopeptide or polypeptide sequence characterised by reversible solubility in response to temperature.

9. The method of claim 8, wherein the temperature-sensitive peptide, oligopeptide or polypeptide sequence comprises at least one copy of the pentapeptide sequence: VPGX'G (SEQ ID NO: 50), wherein X1is any proteinaceous amino acid except proline (Pro).

10. The method of claim 9, wherein the temperature-sensitive peptide, oligopeptide or polypeptide sequence comprises multiple copy repeats of the pentapeptide sequence.

11. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least silver and the step of contacting the fluid material with the peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of KAIHPMR (SEQ ID NO: 1), NSMKHVH (SEQ ID NO: 2), HPITRHK (SEQ ID NO: 3), and RPTKMHR (SEQ ID NO: 4), or a mimetic thereof.

12. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least silver and the step of contacting the fluid material with the peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of H6-SSGGVG-(VPGVG)59-VGVPWP-(GS) / - KAIHPMR (SEQ ID NO: 48) and H6-SSGGVG-(VPGVG)59-VGVPWP-(GS)7-KAIHPMR-GGS-KAIHPMR (SEQ ID NO: 49), or a mimetic thereof.

13. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least gold and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of HSTAPQL (SEQ ID NO: 5), TGFPPYH (SEQ ID NO: 6), LYASRVP (SEQ ID NO: 7), GTGAYDA (SEQ ID NO: 8), KAQTLPA (SEQ ID NO: 9), FNNKHSS (SEQ ID NO: 10), LVTVHYS (SEQ ID NO: 11), TAIRDEL (SEQ ID NO: 12), NHLDDAK (SEQ ID NO: 13), LSLRPIP (SEQ ID NO: 14), SYVGHSP (SEQ ID NO: 15), LPTKPQL (SEQ ID NO: 16) and VAAREYS (SEQ ID NO: 17), or a mimetic thereof.

14. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least magnetite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of GSLGYTG (SEQ ID NO: 20) and WSLGYTG (SEQ ID NO: 21), or a mimetic thereof.

15. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least hematite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), VAVPLHN (SEQ ID NO: 22), HSWRAPY (SEQ ID NO: 23), FNISSSR (SEQ ID NO: 24), NHAQFFK (SEQ ID NO: 25), SHLIAER (SEQ ID NO: 26), and HFHPLES (SEQ ID NO: 27), or a mimetic thereof.

16. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least neodymium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acidsequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), IYAKAPI (SEQ ID NO: 28), LPPYELH (SEQ ID NO: 29), HIGAAYE (SEQ ID NO: 30), and GSPYLFV (SEQ ID NO: 31), or a mimetic thereof.

17. The method of any one of claims 1 to 10, wherein the method is applied to the separation of at least dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), HPSTWHK (SEQ ID NO: 32), FPVVTRN (SEQ ID NO: 33), QSFGPHP (SEQ ID NO: 34), and SYSLNPF (SEQ ID NO: 35), or a mimetic thereof.

18. The method of any one of claims 1 to 10, wherein the method is applied to the separation of one or more of magnetite, hematite, neodymium and dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of the following amino acid sequence:(I) X2SLGYTG (SEQ ID NO: 36); where X2is any proteinaceous amino acid or is null.

19. The method of claim 13, wherein X2is glycine (G), tryptophan (W) or is null.

20. The method of any one of claims 1 to 14, wherein the peptide or mimetic thereof is a multimeric form of a peptide, wherein at least two copies of a particular amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36 are linked by a linker sequence or linker group.

21. The method of any one of claims 1 to 20, wherein the material is an ore, ore preparation, tailings, tailings preparations, tailings dam water and / or settled materials from the bottom of a tailings dam, contaminated soils, wastewater, and waste / recycled materials.

22. The method of claim 21, wherein the material is electronics waste.

23. The method of claim 21, wherein the electronics waste is solar panels / cells and batteries.

24. A peptide comprising or consisting of an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36, or a mimetic thereof.

25. A peptide or mimetic according to claim 24 provided as bound upon a solid support or substrate.

26. A peptide or mimetic according to claim 24 provided in a gas suitable for use in a flotation separation process.

27. A method of assaying a material for the presence of at least one mineral, wherein the method comprises contacting the material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, determining peptide or mimetic thereof that is bound to the desired mineral, wherein the peptide comprises an amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36.

28. The method of claim 27, wherein the peptide or mimetic thereof is provided on the surface of a solid support or substrate.

29. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least silver and the step of contacting the material with the peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of KAIHPMR (SEQ ID NO: 1), NSMKHVH (SEQ ID NO: 2), HPITRHK (SEQ ID NO: 3), and RPTKMHR (SEQ ID NO: 4), or a mimetic thereof.

30. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least gold and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of HSTAPQL (SEQ ID NO: 5), TGFPPYH (SEQ ID NO: 6), LYASRVP (SEQ ID NO: 7), GTGAYDA (SEQ ID NO: 8), KAQTLPA (SEQ ID NO: 9), FNNKHSS (SEQ ID NO: 10), LVTVHYS (SEQ ID NO: 11), TAIRDEL (SEQ ID NO: 12), NHLDDAK (SEQ ID NO: 13), LSLRPIP (SEQ ID NO: 14), SYVGHSP (SEQ ID NO: 15), LPTKPQL (SEQ ID NO: 16) and VAAREYS (SEQ ID NO: 17), or a mimetic thereof.

31. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least magnetite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of GSLGYTG (SEQ ID NO: 20) and WSLGYTG (SEQ ID NO: 21), or a mimetic thereof.

32. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least hematite and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequenceselected from the group consisting of WSLGYTG (SEQ ID NO: 21), VAVPLHN (SEQ ID NO: 22), HSWRAPY (SEQ ID NO: 23), FNISSSR (SEQ ID NO: 24), NHAQFFK (SEQ ID NO: 25), SHLIAER (SEQ ID NO: 26), and HFHPLES (SEQ ID NO: 27), or a mimetic thereof.

33. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least neodymium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), IYAKAPI (SEQ ID NO: 28), LPPYELH (SEQ ID NO: 29), HIGAAYE (SEQ ID NO: 30), and GSPYLFV (SEQ ID NO: 31), or a mimetic thereof.

34. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of at least dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of an amino acid sequence selected from the group consisting of WSLGYTG (SEQ ID NO: 21), HPSTWHK (SEQ ID NO: 32), FPVVTRN (SEQ ID NO: 33), QSFGPHP (SEQ ID NO: 34), and SYSLNPF (SEQ ID NO: 35), or a mimetic thereof.

35. The method of claim 27 or 28, wherein the method is applied to assaying for the presence of one or more of magnetite, hematite, neodymium and dysprosium and the step of contacting the fluid material with a peptide or mimetic thereof comprises contacting the fluid material with a peptide comprising or consisting of the following amino acid sequence:(I) X2SLGYTG (SEQ ID NO: 36); where X2is any proteinaceous amino acid or is null.

36. The method of claim 35, wherein X2is glycine (G), tryptophan (W) or is null.

37. The method of any one of claims 27 to 36, wherein the peptide or mimetic thereof is a multimeric form of a peptide, wherein at least two copies of a particular amino acid sequence selected from the group consisting of those shown as SEQ ID NOs: 1 to 36 are linked by a linker sequence or linker group.

38. The method of any one of claims 27 to 37, wherein the peptide or mimetic thereof is labelled with a detectable label.

39. The method of any one of claims 27 to 38, wherein the material is an ore, ore preparation, tailings, tailings preparations, tailings dam water and / or settled materials from the bottom of a tailings dam, contaminated soils, wastewater, and waste / recycled materials.

40. The method of claim 39, wherein the material is an ore sample.

41. A method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide or mimetic thereof is provided in a multimeric form.

42. A method of assaying a material for the presence of at least one mineral, wherein the method comprises contacting the material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, determining peptide or mimetic thereof that is bound to the desired mineral, wherein the peptide or mimetic thereof is provided in a multimeric form.

43. The method of claim 41 or 42, wherein the multimeric form comprises two copies of the peptide or mimetic thereof joined by a short linker sequence or linker group.

44. The method of claim 43, wherein the two copies of the peptide or mimetic thereof are joined by a flexible GS linker.

45. The method of any one of claims 41 to 44, wherein the peptide is of 7 to 15 amino acids in length.

46. The method of any one of claims 41 to 45, wherein the peptide or mimetic thereof further comprises a hydrophobic moiety or surfactant peptide.

47. The method of claim 46, wherein the hydrophobic moiety is an FF dipeptide moiety.

48. The method of claim 46, wherein the surfactant moiety is MKQLADS-LHQLARQ- VSRLEHA (SEQ ID NO: 53).

49. The method of any one of claims 41 to 48, wherein the peptide or mimetic thereof further comprises a temperature-sensitive peptide, oligopeptide or polypeptide sequence characterised by reversible solubility in response to temperature.

50. The method of claim 49, wherein the temperature-sensitive peptide, oligopeptide or polypeptide sequence comprises at least one copy of the pentapeptide sequence: VPGX'G (SEQ ID NO: 50), wherein X1is any proteinaceous amino acid except proline (Pro).

51. The method of claim 50, wherein the temperature-sensitive peptide, oligopeptide or polypeptide sequence comprises multiple copy repeats of the pentapeptide sequence.

52. A method of separating at least one desired mineral from material comprising same, wherein the method comprises providing said material in a fluid form, contacting said fluid material with a peptide or mimetic thereof which selectively binds to the desired mineral(s) and thereafter, separating mineral(s) bound to the peptide or mimetic thereof from the fluid material, wherein the peptide or mimetic thereof is provided with an elastin-like polypeptide (ELPs) and wherein the said separating of the mineral(s) bound to the peptide or mimetic thereof comprises elevating the temperature of the fluid material to form separable aggregates of mineral(s) bound to the peptide or mimetic thereof.

53. The method of claim 52, wherein the said separating of the mineral(s) bound to the peptide or mimetic thereof comprises elevating the temperature of the fluid material by about 2°C to more than about 25°C.

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