EP3137485A1

EP3137485A1 - Chelator modified magnetic silica nanoparticles, their use and preparation

Info

Publication number: EP3137485A1
Application number: EP15727465.5A
Authority: EP
Inventors: Ana Luísa DANIEL DA SILVA; Rui Miguel PINHEIRO VITORINO; Rui Pedro OLIVEIRA SILVA
Original assignee: Universidade de Aveiro
Current assignee: Universidade de Aveiro
Priority date: 2014-04-28
Filing date: 2015-04-28
Publication date: 2017-03-08
Also published as: WO2015166415A1

Abstract

The present solution relates to magnetic probes/nanoprobes (NP) for the selective enrichment of proteins. Namely, the solution relates to magnetic nanoparticles, which surface is chemically modified with metal ion chelation groups. The use of these magnetic nanoparticles allows the separation of specific proteins from complex mixtures by means of a magnetic field, namely proteins containing metal ions in their composition, such as metalloproteases, and the recovery of metal binding proteins, such as histidine-tagged recombinant proteins. This solution describes a probe for select metalloproteins in a solution comprising at least an inorganic magnetic core particle comprising a paramagnetic, superparamagnetic or ferromagnetic material; wherein such particle is coated with a siliceous coating; wherein the siliceous coating further comprises a plurality of metal ion chelating moieties; wherein the size of the probe is less than 1100 nm. Furthermore, this disclosure also describes a method for producing said probe.

Description

CHELATOR MODIFIED MAGNETIC SILICA NANOPARTICLES,

THEIR USE AND PREPARATION

D E S C R I P T I O N

Technical field

[0001] The present solution relates to magnetic probes/nanoprobes (NP) for the selective enrichment of proteins. More particularly, the solution relates to magnetic nanoparticles in which the surface is chemically modified with metal ion chelation groups. The use of these magnetic nanoparticles allows the separation of specific proteins from complex mixtures by means of a magnetic field, namely proteins containing metal ions in their composition, such as metalloproteases, and the recovery of metal binding proteins, such as histidine-tagged recombinant proteins.

Background Art

[0002] Proteases are a specific group of enzymes that selectively catalyse the breakdown of proteins by hydrolysing peptide bonds. Proteases are crucial for the homeostasis and, therefore, crucial to life, and are involved in numerous physiological processes, namely, digestion, blood coagulation, fertilization, cell differentiation and apoptosis. Matrix metalloproteases (MMPs) are a specific group of proteases, consisting on a family of zinc- containing and calcium-dependent endopeptidases, responsible for the degradation of extracellular matrix and tissue remodeling. Present in diverse biofluids, namely, serum and saliva and, due to their highly catalytic activity, MMPs are usually expressed in minute amounts and their deregulation can lead to pathological conditions.

[0003] Although there are several techniques for the characterization of MMPs and other proteases, such as far-western-blot, ELISA and zymography, they are often limited, lacking specific antibodies for some proteases, being expensive or simply incapable of detecting lower concentrations of proteases, such as those present in some biological traces. The latter is often problematic, as this fact makes them more likely to be masked by other highly abundant proteins. Therefore there is a need for improved methods for the selective enrichment of MMPs from biological fluids and other complex biological mixtures for further analysis. By other side, the production of recombinant proteins in a highly purified and well-characterized form has become a major task for the protein chemist working in the pharmaceutical industry. Thus, several methods have been developed based on immobilized metal affinity chromatography to purify recombinant proteins. One the most widely used affinity-tag systems is based in a short affinity-tag consisting of polyhistidine residues. For instance, histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal which is the ideal for immobilized metal-affinity chromatography.

[0004] Traditional methods for the separation and purification of proteins, namely metalloproteases and histidine-tagged recombinant proteins/peptides consist of using metal-ion affinity chromatography which allow a rapid purification and substantial purity of the product. A widely range support materials have consisted of porous support materials such as agarose, polymethacrylate, polyacrylamide, cellulose, and silica which most of them are commercially available and come in a range of particle and pore sizes. Nevertheless, special emphasis have been given in the last years to magnetic nanoparticles since they offer attractive possibilities for several biomedical and biotechnological applications, namely for bioseparation processes. Due to the intrinsic magnetic properties, magnetic nanoparticles can be easily manipulated by means of an external magnetic field. Additionally, due to nanometric dimensions, magnetic nanoparticles provide high surface to volume ratios and the surface can be subsequently chemically modified to increase the affinity with targeted molecules such as specific proteins. In this context, magnetic assisted separation appears as an attractive method for the selective separation and purification of proteins. For instance Ferreira et al. (Analytic Chemistry 2011, 83, 7035-7043) have developed magnetic nanoparticles with surface functionalized with lectins to selectively capture glycoproteins from human body fluids. However, the use of magnetic nanoparticles for the magnetic assisted enrichment of metalloproteases and histidine-tagged recombinant proteins/peptides is still limited. [0005] For example, Songe et al. (US20060189797 Al) describe magnetic polymer particles comprising cross-linked styrene divinyl benzene polymer, with particle size between 500 nm and 1200 nm, bound to carboxymethylated aspartate ligands an eventually chelating metal ions for the purification of metal binding proteins, histidine- tagged recombinant proteins/peptides and phosphorylated proteins/peptides.

[0006] Hyeon et al. (WO2008010687 Al) describe magnetic nanoparticles of a transition metal such as iron, manganese, nickel, chromium, cobalt and zinc and ions thereof, that will bind selectively to proteins comprising of an amino acid selected from the groups consisting of histidine, asparagine, argentine, cysteine, glutamine, lysine, methionine, proline and trypyophan, for the separation of specific proteins bound to the nanoparticles from biological mixtures, by means of a magnetic field.

[0007] Cheon et al. (WO2010151085 A2) describe zinc containing magnetic nanoparticles coated with binding agents containing functional groups with affinity to the target material (e.g. proteins) for the separation of the target material upon a magnetic field. The improved efficacy of the system in magnetic separation of the target material is due to higher magnetization saturation of the zinc containing particles, when compared to the magnetic particles without zinc used in the invention.

[0008] Ingraham et al. (US20110020894 Al) describe the use of paramagnetic particles with size in the range of micrometres and with the surface functionalized with groups that are used for covalent enzyme attachment, namely epoxide, carboxy, amine, aldehyde, DADPA, or hydrazide-terminated chemical groups for the recovery of biomass degrading enzymes from mixtures of those enzymes and biomass substrates, via magnetic separation.

[0009] Ding et al. (US8147802 B2) describe the surface functionalization of non-magnetic nanoparticles (e.g. Si0₂) with metal ion chelating groups, namely paramagnetic ions such as Gd(lll) for applications in magnetic resonance imaging. [0010] The limitations above-mentioned elucidate the great difficulty of selected and/or remove protein in very low concentrations.

General Description

[0011] The present solution relates to preparation methods and material compositions for the removal of proteins, given if present in very low concentrations, from complex biological mixtures by means of an externally applied magnetic field. In particular, the present solution provides a method for preparing magnetic nanoparticles with surface functionalized with metal ion chelating moieties. The functionalized nanoparticles bind selectively to the metal ion in metalloproteases. Hence, the use of these functionalized nanoparticles is contemplated as a mean to selectively separate metalloproteins and metalloproteases from biological complex mixtures. The use of these magnetic functionalized nanoparticles chelating zinc, nickel, cobalt or other transition metal ions for the purification of histidine-tagged recombinant proteins/peptides is also contemplated in this solution.

[0012] The present solution provides a faster and less expensive probe/nanoprobe and method for the separation and enrichment of specific proteins, often present in biological media in very low concentrations, compared to conventional methods such as metal-ion affinity chromatography. Furthermore different ionic strength buffers can also be used to expand the selective fractionation of proteins from biological samples. Therefore, this disclosure is of great interest in biological and/or pharmaceutical assays, wherein it may be relevant the fast purification of a protein or any other compound comprising a metal ion in its composition and present, for example, in low concentration in body fluids, thereby, promoting the enrichment of specific proteins and minimizing the time required for performing a given assay compared to the existing methods. Due to the high affinity of the nanoprobes to metal ions, the present solution also finds application in the removal of metal ion contaminants from water and industrial effluents, as well as in the development of improved analytical methods for monitoring the quality of water. Furthermore, this disclosure relates to the recovery of proteins present in any solution in very low concentrations.

[0013] None of the above references disclose or suggest a nanoprobe/probe comprising a magnetic core nanoparticle protected by a siliceous coating and containing metal-ion chelating groups at the surface of the nanoprobe, for the selective enrichment of specific proteins via the magnetic separation.

[0014] A metalloprotein is a protein comprising a metal ion in the catalytic centre.

[0015] A metalloproteinase is a metalloprotein with proteolytic activity and responsible for catalysing the breakdown of proteins by hydrolysing the peptide bonds of the proteins.

[0016] A paramagnetic material is any material presenting at least one unpaired electrons and susceptible to present magnetization when attracted by an external magnetic field.

[0017] A ferromagnetic material is any material presenting at least one unpaired electrons and possessing magnetization even in the absence of an external magnetic field.

[0018] A superparamagnetic material is any material presenting at least one unpaired electrons and sufficiently small, namely nanoparticles, and wherein magnetization may randomly change direction under the influence of temperature.

[0019] A metal-ion chelating agent forms one or more coordinate bonds with metal ions of, for example, a metalloprotease. A biological sample may be a sample of water, waste water, human saliva, plasma, blood, urine, food, among others.

[0020] An aspect of the present invention is relate to a probe/nanoprobe for select metalloproteins in a solution comprising :

at least an inorganic magnetic core particle comprising a paramagnetic, superparamagnetic or ferromagnetic material;

wherein such particle is coated with a siliceous coating; wherein the siliceous coating further comprises a plurality of metal ion chelating moieties;

wherein the size of the probe is less than 1100 nm, preferably less than 1000 nm, more preferably 20-400 nm, even more preferably 10-75 nm.

[0021] In an embodiment of the probe of the present disclosure the size of the inorganic magnetic core of the probe may be less than 1000 nm, preferably 10-300 nm, between 1 - 150 nm.

[0022] In an embodiment of the probe of the present disclosure the material of the core particle may be selected from the following list consisting of: iron, nickel, cobalt, zinc, alloys of iron, nickel, cobalt, zinc, their oxides, or mixtures thereof. Ina a preferred embodiment the material of the core particle may be iron oxide, magnetite, maghemite or mixture thereof.

[0023] In an embodiment of the probe of the present disclosure the siliceous coating may comprise silanol groups.

[0024] In an embodiment of the probe of the present disclosure the thickness of the siliceous coating may be between 0.5 - 100 nm; preferably between 2 - 25 nm, more preferably between 3 - 15 nm.

[0025] The characterization of the size of the particle, and thickness may be measured by many methods available in the prior art, in particular by scanning electron microscopy (SEM) and or transmission electron microscopy (TEM).

[0026] In an embodiment of the probe of the present disclosure the metal-ion chelating may be organosilane compounds containing in its structure N- (silylpropyl)ethylenediamine triacetic acid, among others.

[0027] In an embodiment of the probe of the present disclosure the metal-ion chelating group may be a organosilane selected from the following list: N- (trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt, N- (triethoxysilylpropyl)ethylenediamine triacetic acid salt or a mixture thereof, among others.

[0028] In an embodiment of the probe of the present disclosure:

the metal-ion chelating is N-(trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt;

the inorganic magnetic core is iron, or iron oxide, magnetite, maghemite or a mixture thereof.

[0029] Another aspect of the present invention is relate to a method for producing the probe described in the present disclosure claims comprising the following steps:

synthetizing a inorganic magnetic core particle paramagnetic, superparamagnetic or ferromagnetic material under N2 atmosphere;

coating the inorganic particle with a alkoxysilane moiety by hydrolysis and

condensation;

adding a metal ion chelating moiety by reacting the alkoxysilane moiety with an organosilane comprising N-(silylpropyl)ethylenediamine triacetic acid, N- (trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt, N- (triethoxysilylpropyl)ethylenediamine triacetic acid salt or a mixture thereof wherein addition of the ion chelating moieties is conducted

at temperatures between 20 - 95 °C, more preferably between 70 - 90 °C;

for 1 min - 48 h, more preferably between 18 - 24 hours

in acidic conditions.

[0030] In an embodiment of the method the alkoxysilane moiety may be selected from the following list: tetraethyl orthosilicate, Tetramethyl orthosilicate (TMOS), Tetrakis(2- hydroxyethyl)orthosilicate (THEOS), or their mixtures thereof, among others.

[0031] Another aspect of the present invention is relate use of the probe of the disclosure subject matter for the identification, separation and enrichment of proteins in biological samples. Preferably wherein such biological sample is water, human saliva, waste water, blood, urine, food, among others.

[0032] The present solution also relates to the preparation of metal ion chelating magnetic nanoprobes/probes, wherein said nanoprobes/probes comprise a core particle that can be attracted by an externally applied magnetic field and the coating of said magnetic core may be made with a shell of a siliceous material providing silanol groups at the surface, and characterized by the reaction of said coated magnetic particle with an organosilane containing in its structure N-(silylpropyl)ethylenediamine triacetic acid, in particular wherein the organosilane may be N-(trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt (EDTA-TMS), N-(triethoxysilylpropyl)ethylenediamine triacetic acid salt or a mixture of both. Furthermore, the reaction may be performed in acidic aqueous medium at temperatures between room temperature and 95 °C, preferably between 70 and 90 °C and carried out between 1 minute and 48 hours, more preferably between 18 and 24 hours.

[0033] In an embodiment, the core particle may be composed of any inorganic material that could be attracted by an external magnetic field including metals such as iron, nickel, cobalt, zinc, alloys of these metals and magnetic iron oxides, in particular, the core particle may be composed by magnetite, maghemite or a mixture of both phases and the core particle is coated by a shell of amorphous silica.

[0034] In an embodiment, the method for the selective separation of metalloproteases wherein the magnetic nanoprobes obtained using the procedure described above may be dispersed in the medium containing metalloproteases, in particular for 30 minutes and then separated upon the exposure to an external magnetic field.

[0035] In an embodiment, the method for the separation of histidine-tagged proteins and peptides wherein the chelating groups of the magnetic nanoprobes obtained using the procedure previously described are charged with Ni²⁺ ions or other metal transition ions with high affinity to histidine chosen from the group Zn²⁺, Cu²⁺, and Co²⁺, prior the contact with the solutions containing histidine tagged proteins or histidine tagged peptides and then are separated upon the exposure to an external magnetic field.

[0036] Throughout the description and claims the word "comprise" and va riations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Brief Description of the Drawings

[0037] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0038] Figure 1 shows a step-by-step synthesis process of magnetic nanoparticles coated with a siliceous material, whose surface was chemically modified with EDTA-TMS.

[0039] Figure 2 shows a scheme of the reaction of surface modification of coated magnetic particles using EDTA-TMS for the attachment of metal-ion chelator ligands at the surface.

[0040] Figure 3 shows the powder XRD diffractogram of magnetite nanoparticles with an average size of 12 nm.

[0041] Figure 4A shows a TEM image of magnetite nanoparticles with an average size of 12 nm.

[0042] Figure 4B shows the histogram of the diameter of the magnetite particles with an average size of 12 nm, obtained from TEM images. [0043] Figure 5 shows the magnetization curve of superparamagnetic Fe₃0₄ and Fe₃0₄@Si02-CH as function of the magnetic field at 300 K. The inset shows loop in more detail.

[0044] Figure 6 shows a TEM image of magnetite nanoparticles with an average size of 12 nm coated with a layer of amorphous silica.

[0045] Figure 7 shows the ATR-FTIR spectra of the EDTA-TMS, the magnetite core particles (Fe₃0₄) with an average size of 12 nm, those magnetite core particles coated with a silica shell (Fe₃0₄@SiC>2) and of those particles after surface modification with EDTA-TMS (Fe₃0₄@Si0₂-CH) at 70 °C.

[0046] Figure 8 shows the powder XRD diffractogram of magnetite nanoparticles with a n average size of 50 nm.

[0047] Figure 9A shows a SEM image of magnetite nanoparticles with an average size of 50 nm.

[0048] Figure 9B shows the histogram of the diameter of the magnetite particles with an average size of 50 nm, obtained from SEM images.

[0049] Figure 10 shows the magnetization curve of ferromagnetic particles Fe₃0₄ and Fe₃0₄@Si0₂-CH as function of the magnetic field at 300 K. The inset shows loop in more detail.

[0050] Figure 11 shows a TEM image of magnetite nanoparticles with an average size of 50 nm coated with a layer of amorphous silica.

[0051] Figure 12 shows the ATR-FTIR spectra of the magnetite core particles (Fe₃0₄) with an average size of 50 nm, those magnetite core particles coated with a silica shell (Fe₃0₄@Si0₂) and those particles after surface modification with EDTA-TMS (Fe₃0₄@Si0₂- CH) at 70 °C. [0052] Figure 13 shows the zymography profile from incubation of Fe30₄@Si02-CH NPs with human saliva diluted 1:10 in MES buffer; A- Control; B- Assay using 50 nm particles; C- Assay using 12 nm particles.

[0053] Figure 14A shows the SDS-PAGE (12.5%) profile of the eluted fractions obtained by incubation of the 50 nm magnetic particles after Ni²⁺ chelation, with the cell extract containing the his-tag protein, diluted 1:10 in MES buffer, and respective control.

[0054] Figure 14B shows the Western-Blot profile of the eluted fractions obtained by incubation of the 50 nm magnetic particles after Ni²⁺ chelation, with the cell extract containing the his-tag protein, diluted 1:10 in MES buffer, and respective control.

Detailed Description

[0055] The solution relates to magnetic nanoparticles with surface functionalized with metal ion chelator ligands, a method for producing such particles and applications of those particles in the separation and enrichment of proteins, by means of an externally applied magnetic field.

[0056] The method for the production of the particles is illustrated in Figure 1 and involves: (A) a magnetic core particle, (B) the coating of the core particle with a siliceous material providing silanol groups at the surface and, (C) the modification of the surface of the coated particles for the incorporation of metal ion chelator ligands.

[0057] Magnetic core particles contemplated for use in the present solutions include but are not limited to those described herein. Any paramagnetic, superparamagnetic or ferromagnetic particle for providing separation upon an external magnetic field and compatible with aqueous medium are contemplated for use in the present solution. The average size of the magnetic core particles may be preferably within the nanometer range, in particular less than 1000 nm, more preferably between 1 - 150 nm. The particles may be comprised by any type of material that exhibits magnetic properties, for example metals such as iron, nickel, cobalt, alloys of these metals and magnetic iron oxides. [0058] The growing of a siliceous shell around the magnetic core nanoparticles is performed by hydrolysis and condensation of alkoxysilanes, including but not limited to the tetraethyl orthosilicate (TEOS), in a homogeneous alcoholic medium, using a base as catalyst, provided that silanol groups are available at the surface of the coated particles.

[0059] The thickness of the siliceous shell may be preferably between 0.5 and 100 nm, provided that the coated particles are magnetic and easily attracted by an externa l magnetic field. Preferably thickness of the siliceous shell is between 2 and 25 nm, most preferably between 3 and 15 nm.

[0060] The surface of the coated particles is modified with metal ion chelator ligands, by reaction with an organosilane containing in its structure N-(silylpropyl)ethylenediamine triacetic acid, namely the organosilane N-(trimethoxysilylpropyl) ethylene diamine triacetic acid trisodium salt (EDTA-TMS), in aqueous acidic conditions, as depicted in Figure 2. The reaction can be performed, in particular, at temperatures between room temperature up to 95 °C, most preferably between 70 and 90 °C. The reaction time may be between 1 minute and 48 hours, most preferably between 18 and 24 hours.

[0061] The present solution also provides applications of the magnetic nanoprobes for the selective enrichment of metalloproteases and histidine tagged proteins. The magnetic nanoprobes are dispersed in the mixture containing metalloproteases. The amine and carboxylate groups of the chelating ligands attached at the surface of the particles chelate with a number of metal ions present in metalloproteases. The application of an external magnetic field to the magnetic particles chelating cations from proteases provides a mean for the selective separation of those proteases from the surrounding medium. For applications in the recovery of histidine-tagged proteins, the functional groups at the surface of the magnetic nanoprobes are first chelating metal transition ions, including but not limited to Zn(ll), Cu(ll), Ni(ll) and Co(ll), with high affinity to histidine. The magnetic particles chelating the metal ions are then dispersed in biological mixtures containing histidine-tagged proteins and those proteins are separated from the mixture by means of an externally applied magnetic field. [0062] The use of the magnetic nanoprobes described in the present solution for the selective enrichment of specific proteins presents several advantages when compared to the existing methods, based on metal-ion affinity chromatography. Namely, the present solution provides a faster method of separation, less expensive than those using chromatography columns and requiring reduced amount of magnetic nanoprobes due to the high specific surface area arising from the nanometric dimensions of the particles and due to the significant amount of metal-ion chelator ligands attached to the surface of the particles. Furthermore, the growth of a siliceous shell around the magnetic core particle provides better stability and prevents the ion leaching from the particles, when compared with solutions where the magnetic core is directly in contact with the biological medium.

Examples

[0063] The following examples are presented herein for illustration only and should not be construed as limiting the solution in any way.

[0064] Unless otherwise noted, all chemicals were used as received. Iron (III) chloride hexahydrated (FeCI₃.6H₂0, >99%), iron (II) chloride tetrahydrated (FeCI₂.4H₂0, >99%), tetraethyl orthosilicate (TEOS, 99.99%), Sodium citrate dihydrate (99%), triethylamine (99%), iron (II) sulphate heptahydrated (99%), 2-(/V-morpholino)ethanesulfonic acid (low moisture MES, 99%), 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES, 99.5%), tetraethylammonium bicarbonate (TEAB), gelatin from porcine skin (99%), ammonium persulfate (APS, >98%), Ν,Ν,Ν',Ν'-tetramethylethylenediamine (TEMED 99%), glycerol (99%), triton X-100, ethylenediaminetetraacetic acid disodium salt dihydrate (99%), zinc chloride (99.99%) and calcium chloride (anhydrous, 99.99%) were purchased from Sigma-Aldrich. Nitric acid (HN0₃, 25%), Bromophenol Blue and glacial acetic acid were acquired from Panreac and used as supplied. Ammonia (NH4OH) (25%, NH₃) and zinc sulphate (p. a.) were obtained from Riedel-de-Haen. Coomassie Brilliant Blue G-250 (p. a.) and hydrochloric acid (HCI, 37%) were purchased to Fluka. EDTA-TMS (trimethoxysilylpropyl)ethylenediamine triacetic acid sodium salt (45% in water) was acquired to Gelest. Sodium dodecyl sulfate (SDS, 99%) was bought from USB and acrilamid (40%) and bis-acrilamid (2%) were purchased from BioRad. Sodium chloride (99.9%) and methanol (p. a) were obtained from Analar Normapur and Tris (99.8%) from Plus One.

Example 1 - Preparation of superparamagnetic nanoprobes with metal ion chelator ligands bound at the surface

[0065] In this example, the preparation and characterization of representative nanoprobes with chelating moieties at the surface and superparamagnetic properties is described.

[0066] Synthesis of the magnetic iron oxide core. Magnetic iron oxide NPs with an average size of 12 nm were synthesized using the co-precipitation method, as follows. Typically, 4.43 g FeCI₃.6H₂0 and 1.625 g of FeCI₂.4H₂0 were dissolved in 190 ml of distilled water at room temperature, under N₂ atmosphere and mechanical stirring. Afterwards, 10 ml of ammonia were added to the solution and stirred for 10 min. The final mixture displayed a black coloration that suggests the formation of the iron oxide magnetite (Fe₃0₄) following the reaction:

2 Fe³⁺ (aq) + Fe²⁺ (aq) + 8 OH^" ► Fe₃0₄(s) + 4 H₂0

[0067] The nanoparticles were magnetically separated and washed five times with distilled water. Powder X-ray diffraction (XRD) was conducted on the dried samples. An example of the XRD diffractogram is shown in Figure 3 and confirms that magnetite is the main crystalline phase on the sample. The particle size distribution was assessed by Transmission electron microscopy (TEM) as shown in Figure 4A and the calculated average particle size was 11.8 ± 3.2 nm as seen in the particle size histogram (Figure 4B). The dependence of the magnetization with the applied magnetic field at 300 K show no hysteresis (Figure 5), confirming that the magnetic core particles were superparamagnetic. Prior to silica encapsulation, the magnetic core particles were stabilized with citrate ions to prevent agglomeration. Thus, the particles were washed twice with an aqueous solution of HN0₃ 2 M, magnetically separated, washed with distilled water and the pH was set at 2.5 (25 °C). Then, sodium citrate (5 ml, 0.5 M) was added to the particles suspension (200 mL) and the solution was left stirring for lh at room temperature. Afterwards, the NPs were magnetically recovered, washed thoroughly with distilled water and freeze-dried.

[0068] Coating of the magnetic core with a shell of amorphous silica. Silica coating of the particles-12 nm was performed by hydrolysis of TEOS in alkaline conditions using triethylamine as catalyst. Typically, a suspension of the particles (100 mg) in 18 mL of distilled water was sonicated for 10 min to prevent particle aggregation followed by the addition of 1 mL of TEOS (30% v/v in ethanol) and 0.1 mL de triethylamine. The reaction was performed under sonication (Horn Sonics, Vibracell) for 15 min at room temperature and the particles magnetically recovered as a black powder hereafter designated as Fe₃0₄@Si0₂, and washed thoroughly with distilled water and freeze-dried. The formation of a coating around the magnetic core with an average thickness of 4.9 ± 1.9 nm was confirmed by TEM analysis (Figure 6) and the chemical nature of the coating was assessed by FTIR spectroscopy. The FTIR spectrum (Figure 7) of the particles after coating show the appearance of absorption bands with maxima at 950 cm ¹ and 1053 cm⁴, assigned to Si- O-Fe and to Si-O-Si asymmetric stretching, respectively, in agreement with the formation of a silica shell.

[0069] Functionalization of the surface of the silica coated nanoparticle with chelating moieties. In order to introduce the chelating groups at the surface of the silica coated NPs, these were modified with EDTA-TMS. In a typical procedure, 80 mg of Fe₃0₄@Si02 were dispersed in 2.5 ml of ddH₂0 and then added to a solution comprising EDTA-TMS (1 ml; 1.23 mmol) and double distillated water -ddH₂0 (2 ml). Then, a glacial acetic acid was added and the suspension was mechanically stirred under reflux. The amount of acetic acid (50 and 100 μΙ), the reaction temperature (70 and 90 °C) and reaction time (18 and 24h) was varied. After reaction, the particles containing chelating groups at the surface (hereafter designated as Fe₃0₄@Si0₂-CH) were magnetically recovered and washed with distilled water and freeze-dried. The amount of EDTA-TMS attached to the surface of the final particles was estimated from nitrogen content, determined by elemental analysis, and varied up to 0.47 mmol/g particles (Table 1). The final particles were superparamagnetic, with no histeresis loop and zero remanence and coercive field (Figure 5). The magnetization saturation (Ms) of the particles functonalized at 70 °C was 64 emu/g at 300K. The specific surface area of those particles was assessed by nitrogen adsorption Brunauer-Emmett-Teller (BET) measurements and was 91 m²/g-

[0070] Table 1. Characteristics of the superparamagnetic particles before and after surface functionalization with metal ion chelators.

Example 2 - Preparation of ferromagnetic nanoprobes with metal ion chelator ligands bound at the surface

[0071] In this example, the preparation and characterization of representative nanoprobes with metal ion chelating moieties at the surface and ferromagnetic properties is described.

[0072] Synthesis of the magnetic iron oxide core: magnetic iron oxide nanoparticles with an average size of 50 nm were synthesized by oxidative hydrolysis of iron(ll) sulphate in alkaline conditions, as follows. In a round flask, 200 mL of milli-Q water was deoxygenated with N₂ under vigorous stirring during 2 hours. Then, 25 mL of deoxygenated water was added to a 250 mL round flask and 1.899 g and 1.519 g of KOH and KN0₃, respectively, were added. The resulting mixture was heated at 60 °C with bubbling N₂ and mechanically stirred at 500 rpm. After salt dissolution, 25 mL of an aqueous solution containing 4.745g of FeS0₄.7H₂0 was added drop-by-drop and the stirring was increased to 700 rpm. The resulting solution presented a dark-green color after complete addition of the Fe²⁺ salt. The solution was left to react for 30 minutes. After reaction, the round flask was transferred to a hot oil bath (90 °C) and left under N₂ with no stirring during 4 hours. Finally, the resulting black powder was washed several times with deoxygenated water and ethanol. After washing, particles were dried by evaporating the solvent. The XRD diffractogram of dried sample is shown in Figure 8 and confirms that magnetite is the main crystalline phase on the sample. The particle size distribution was assessed by Transmission electron microscopy (TEM) as shown in Figure 9A and Figure 9B, and the calculated average particle size was 52.6 ± 8.3 nm. The dependence of the magnetization with the applied magnetic field at 300 K showed hysteresis (Figure 10), with small coercivity and remanence, thus indicating that the magnetic core particles were ferromagnetic.

[0073] Coating of the magnetic core with a shell of amorphous silica: silica coating of the particles-50 nm was carried out by hydrolysis of TEOS in alcoholic conditions using ammonia as catalyst. Typically 50 mg of magnetic particles were added to 40 ml ethanol. The suspension was left to sonicate for 10 min, allowing for the particles to be highly dispersed in the solution. After, 100 μΙ of TEOS and 3 ml of ammonia (25%) were added to the solution which was left for 2 hours at room temperature under sonication. The resulting particles were washed with distilled water and ethanol and then were left to dry by solvent evaporation. TEM images (Figure 11) confirm the formation of a layer 10.5 ± 2.2 nm thickness around the magnetic core, and the FTIR spectrum (Figure 12) shows the appearance of the bands centred at 950 cm ¹ and 1053 cm ¹, assigned to Si-O-Fe and to Si- O-Si asymmetric stretching, respectively, in agreement with the formation of a silica shell.

[0074] Functionalization of the surface of the silica coated nanoparticle with chelating moieties: silica coated particles were reacted with EDTA-TMS. Thus, in a typical procedure, 80 mg of Fe30₄@Si0₂ were dispersed in 2.5 ml of ddH₂0 and then added to a solution comprising EDTA-TMS (1 ml; 1.23 mmol) and ddH₂0 (2 ml). Then, a glacial acetic acid (500 μΙ) was added and the suspension was mechanically stirred under reflux. The reaction temperature (70 and 90 °C) and reaction time (18 and 24h) was varied. After reaction, the particles containing chelating groups at the surface (Fe30₄@Si0₂-CH) were magnetically recovered and washed with distilled water and freeze-dried. The amount of EDTA-TMS attached to the surface of the final particles was estimated from nitrogen content, determined by elemental analysis, and varied up to 0.112 mmol/g particles (Table 2). The particles functionalized at 70^C were ferromagnetic with small remanence (8.2 emu/g) and coercivity (160 Oe)(Figure 10) and magnetization saturation (Ms) of 40 emu/g at 300K. The specific surface area of those particles was assessed by nitrogen adsorption Brunauer-Emmett-Teller (BET) measurements and was 17.9 m²/g-

[0075] Table 2. Characteristics of the ferromagnetic particles before and after surface functionalization with metal ion chelators.

Example 3 - Application of the magnetic nanoprobes on the selective enrichment of metalloproteases from saliva

[0076] The magnetic nanoparticles containing chelating moieties at the surface (here designated as Fe₃0₄@Si0₂-CH) were tested as a means of selectively capture metalloproteases (MMPs) present in human saliva. Superparamagnetic and ferromagnetic Fe₃0₄@Si0₂-CH particles with magnetic core of 12 and 50 nm prepared as in the examples 1 and 2, respectively, with surface functionalization carried out at 70 °C, were used in incubation.

[0077] Saliva samples were obtained from healthy human donors. To eliminate inter- individual contributions, analyses were carried out with pools of proteins from different individuals. Unstimulated saliva was collective by passive drooling, and kept in ice. After collection, saliva was centrifuged at 12000 g and 4 °C for 30 min to remove any particulates present. Supernatant was collected and stored at -70 °C until further utilization.

[0078] The ability of Fe30₄@Si02-CH particles to specifically uptake metalloproteases was evaluated by incubating accurately weighted 0.3 mg of particles in 500 μΙ (final volume) of saliva diluted 1:10 in MES buffer.

[0079] After incubation, the particles were removed magnetically using a DynaMag^Tm - Spin Magnet from Invitrogen - Life Techonologies for about 1 minute and washed with the MES buffer three consecutive times, aiming at the removal of proteins unspecifically adsorbed to the surface of the nanoparticles. The captured proteins were analyzed by zymography of polyacrylamide gel copolymerized with gelatin, in order to confirm the specificity of the nanoprobes for capturing metalloproteases.

[0080] Besides common zymography, this assay was also performed after addition of Zn²⁺ to the loading buffer to evaluate any competition of these cations for the chelating sites and consequent release of the MMPs to the medium. Additionally, a zymographic assay was performed by incubating a fraction of the gel in the development buffer supplemented with 10 mM EDTA. The aim of this assay was to validate if the gelatinolytic activity observed in the zymography was due to MMPs activity.

[0081] Gelatin was selected as substrate for zymographic assays since metalloproteases have ability to degrade it. Briefly, protein-linked particles were incubated with the loading buffer (233 μΙ milli-Q water, 500 μΙ 10% SDS, 66.7 μΙ Tris-HCI 0.5 M pH 6.8, 200 μΙ glycerol and bromophenol powders) or with zinc-modified loading buffer (modified with zinc sulfate 25 mM), in 1:1 (v/v) proportion and left to react during 15 min at 37^ C. Furthermore, proteins were separated by 12.5% SDS-PAGE gel copolymerized with 0.1% gelatin. After electrophoresis, gels were washed with a 2.5% Triton X-100 solution and incubated overnight at 37 °C in development buffer (50 mM Tris-HCI, pH 7.6, 10 mM CaCI₂ and 10 mM ZnCI₂) or in development buffer supplemented with 10 mM EDTA to determine the presence of metalloproteases. Then, the gel was stained using a 0.5 % w/v coomassie brilliant blue (CBB) G250 solution overnight under mechanical stirring. Finally, gels were unstained using a methanol (40%) and acetic acid (10%) solution. I mages of the different gels were acquired using a X-ray films were scanned in Molecular Imager Gel Doc Xr+ System and analyzed with Quantity One software v. 4.6.9 (Bio-Rad). The results of the zymographic assays are shown in the Figure 13.

[0082] Both assays with 12 nm and 50 nm magnetic nanoprobes show gelatinolytic activity, this being more intense for the later. When Zn²⁺ ions were added to the loading buffer, only a small increase of gelatinolytic activity was observed. Lastly, when incubated with 10 mM EDTA in the development buffer, no gelatinolytic activity was observed. Since EDTA is a well-known inhibitor of metalloproteases, these results validate the enrichment of metalloproteases using the magnetic nanoprobes. These results confirm the capabi lity of the nanoparticles functionalized with chelating moieties to specifically enrich human saliva samples in metalloproteases, and, in particular, MMP-9.

Example 4. Application of the magnetic nanoprobes on the selective separation of histidine-tagged proteins.

[0083] The magnetic nanoparticles containing chelating moieties at the surface ( here designated as Fe304@Si02-CH) were tested as a mean of selectively separate histidine- tagged recombinant proteins from biological mixtures comprising an Escherichia Coli extract with a recombinant protein the Synphilin-1A tagged with a histidine chain in the N-terminal. Ferromagnetic Fe304@Si02-CH particles prepared as in the example 2, with surface functionalization carried out at 70 °C, were tested.

[0084] The ferromagnetic Fe304@Si02-CH particles were incubated during 2 hours with nickel sulfate (NiS0₄.6H₂0, 1 M). Afterwards the particles were washed using MES (2-(N- morpholino)ethanesulfonic acid, 0.01 M) buffer. Then the particles (1 mg) were incubated with the cell extract diluted 1:10 in MES buffer (0.1 M), up to a final volume of 500 μί. The incubation was carried out for 2h, under mechanical stirring. After incubation, the particles were magnetically separated from the mixture and washed 3 times with MES buffer and then the His-Tag proteins were eluted by incubation of the particles with imidazole solution (1 M) during 15 minutes.

[0085] The eluted fractions were analyzed by sodium dodecyl sulfate 12.5% polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, run at 150 V until completion, the gel was left in a solution of methanol (40%) and acetic acid (10%) for 1 h for fixation and was then stained overnight in a colloidal coomassie solution. Afterwards, the gel was unstained using a solution of methanol (25%). The SDS-PAGE results are shown in Figure 14A.

[0086] The eluted fractions were also analyzed by Western-Blot (using a Mouse anti-His antibody). Figure 14B shows the western blot of the original extract (control - approximately 30 μg total protein) and of the eluted fractions obtained by incubation of the magnetic particles after Ni²⁺ chelation. In this assay the Anti-His (from mouse) antibody was used as a primary antibody and an anti-mouse was used as secondary antibody. The comparison of the Western-Blot profiles (Figure 14B) with coomassie profile (Figure 14A) allow to conclude that the Fe304@Si02-CH particles incubated with Ni²⁺ display high capacity to interact specifically with the His-tag proteins and can be used for their enrichment in a short time, when compared with traditional columns.

[0087] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0088] The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S

1. A probe for select metalloproteins in a solution comprising:

wherein such particle is coated with a siliceous coating;

wherein the siliceous coating further comprises a plurality of metal ion chelating moieties;

wherein the size of the probe is less than 1100 nm, preferably less than 1000 nm.

2. Probe according to the previous claim wherein the size of the inorganic magnetic core is less than 1000 nm, preferably 1 - 150 nm.

3. Probe according to the previous claims wherein the material of the core particle is selected from the following list consisting of: iron, nickel, cobalt, zinc, alloys of iron, nickel, cobalt, zinc, their oxides, or mixtures thereof.

4. Probe according to claim 3 wherein the material of the core particle is iron oxide, magnetite, maghemite or mixture thereof.

5. Probe according to the previous claims wherein the siliceous coating comprise silanol groups.

6. Probe according to the previous claims wherein the thickness of the siliceous coating is between 0.5 - 100 nm; preferably between 3 - 15 nm.

7. Probe according to the previous claims wherein the metal-ion chelating is organosilane compounds containing in its structure N-(silylpropyl)ethylenediamine triacetic acid.

8. Probe according to the previous claims wherein the metal-ion chelating group is a organosilane selected from the following list: N- (trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt, N- (triethoxysilylpropyl)ethylenediamine triacetic acid salt or a mixture thereof.

9. Probe according to the previous claims wherein

10. A method for producing the probe described in any of the previous claims comprising the following steps:

synthetizing a inorganic magnetic core particle paramagnetic, superparamagnetic or ferromagnetic material under N₂ atmosphere;

coating the inorganic particle with a alkoxysilane moiety by hydrolysis and condensation;

adding a metal ion chelating moiety by reacting the alkoxysilane moiety with an organosilane comprising N-(silylpropyl)ethylenediamine triacetic acid, N- (trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium salt, N- (triethoxysilylpropyl)ethylenediamine triacetic acid salt or a mixture thereof;

wherein addition of the ion chelating moieties is conducted;

at temperatures between 20 - 95 °C, more preferably between 70 - 90 °C;

for 1 min - 48 h, more preferably between 18 - 24 hours;

in acidic conditions.

11. Method according to the previous claim wherein the alkoxysilane moiety is selected from the following list: tetraethyl orthosilicate, Tetramethyl orthosilicate , Tetrakis(2-hydroxyethyl)orthosilicate , or their mixtures thereof.

12. Use of the probe described in any of the previous claims for the identification, separation and enrichment of proteins in biological samples.

13. Use of the probe described in the previous claims wherein such biological sample is water, human saliva, waste water, blood, urine, food.