SURFACE MODIFIED PARTICLES

Abstract

Surface modified particles have a core, an inner shell and an outer shell. The core is formed of silica or is hollow, the inner shell is formed by a layer of metal, and the outer shell is formed by a biocompatible polymer brush. The particles allow for direct optical detection of biomolecules such as nucleic acids, proteins, polysaccharides and glycoproteins in biological samples.

Claims

1. Surface-modified particles, characterized in that they comprise a core, an inner shell and an outer shell, wherein the core is formed of silica or the core is hollow; and the core has a diameter d.sub.1 in the range of 20 nm to 1 .Math.m, the inner shell consists of a layer of metal M, said layer having a thickness d.sub.2 in the range of 2 to 60 nm, the outer shell has a thickness d.sub.3 in the range of 2 to 200 nm, and the outer shell consists of a layer of a polymer of the general formula ##STR00022## wherein x = 2 to 50, y = 5 to 5000, z = 0 to 2000, z/y = 0 to 0.4; R are the same or different on each occurence, wherein each R is independently selected from the group consisting of —(CH.sub.2).sub.n—C≡CH, —(CH.sub.2).sub.m—N.sub.3, —(CH.sub.2).sub.n—NH.sub.2, —(CH.sub.2).sub.n—COOH, ##STR00023## ##STR00024## wherein n = 1 to 4, m = 2 to 5; R.sup.1 is selected from the group consisting of ##STR00025## ##STR00026## and a fluorophore, wherein p = 0 to 24, q = 2 or 3, r = 0 to 24; R.sup.2 is selected from the group consisting of ##STR00027## ##STR00028## fluorophore, wherein p =0 to 24, q = 2 or 3, r= 0 to 24, t = 1 to 4, u = 0 or 1; wherein when R.sup.1 is ##STR00029## the biotin moiety is optionally conjugated to a biotin-binding protein such as neutravidin, streptavidin, or avidin, via formation of a non-covalent attachment between biotin and the biotin-binding protein; and wherein when R.sup.1 is ##STR00030## and it is conjugated to the biotin-binding protein, a biotin-modified biomolecule is optionally non-covalently attached to the biotin-binding protein via formation of a non-covalent attachment between the biotin-binding protein and the biotin moiety of the biotin-modified biomolecule; wherein the polymer of the general formula II is attached to the surface of the inner shell by means of its sulphur atoms forming an M-S bond with the metal atoms of the inner shell, as depicted by the dashed bonds in the formula II.

2. Surface-modified particles according to claim 1, characterized in that the inner shell has a thickness d.sub.2 in the range of 3 to 25 nm, more preferably 5 to 20 nm; and/or the outer shell has a thickness d.sub.3 in the range of 5 to 100 nm, more preferably 10 to 60 nm.

3. A process for the preparation of surface-modified particles according to claim 1, wherein particles comprising a core and an inner shell are reacted with a compound of formula III ##STR00031## wherein x is as defined in claim 1, wherein the compound of formula III is optionally in a mixture with lipoic acid in a molar ratio of lipoic acid: compound of formula III = 2:1 to 6:1, preferably 4:1, and the product is subsequently contacted with monomer of formula IV under free radical polymerization conditions ##STR00032## wherein the monomer of formula IV optionally contains an admixture of 0 to 40 mol% of monomer of formula V ##STR00033## wherein R is as defined in claim 1, to form a polymer of formula II ##STR00034## wherein R, x, y, z are as defined in claim 1, attached to the inner shell, thereby forming the outer shell.

4. The process for the preparation of surface-modified particles according to claim 1, wherein a compound of formula IIA ##STR00035## is reacted with polymer of formula IIB ##STR00036## to form polymer of formula IIC ##STR00037## wherein R, x, y, z are as defined in claim 1, and the polymer of formula IIC is subsequently reacted in an aqueous medium, optionally in a mixture with lipoic acid in a molar ratio of lipoic acid : polymer of formula IIC = 2:1 to 6:1, preferably 4:1, with particles comprising a core and an inner shell as defined in claim 1, to form particles with polymer II ##STR00038## bound to the inner shell and forming the outer shell.

5. A process according to claim 3, wherein the particles comprising the core and the inner shell contain gold inner shell and are prepared by the following steps: in a first step, silica cores are modified by reaction with trialkoxysilane derivatives of formula R.sup.4Si(R.sup.3).sub.3, wherein R.sup.4 is selected from C.sub.2-C.sub.4 alkyl terminally substituted with mercapto or amino group, and R.sup.3 are selected from the group consisting of —OCH.sub.3 and —OCH.sub.2CH.sub.3, in a second step, gold nanoparticles with a diameter of less than 5 nm are bound to the thus modified core particles, in a third step, the resulting particles, formed by a core with bound gold nanoparticles, are reacted with [AuCl.sub.4.sup.-] in the presence of a reducing agent, thus forming an inner shell.

6. The process according to claim 5, wherein in the first step the trialkoxysilane derivatives are selected from the group consisting of (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, and (3-aminopropyl)triethoxysilane.

7. The process according to claim 5, wherein in the third step, the reducing agent is selected from the group consisting of carbon monoxide, hydroxylamine, hydrazine, methylhydrazine, ascorbic acid, formaldehyde and acetaldehyde.

8. The process according to claim 3, wherein the particles comprising the core and the inner shell contain gold inner shell and are prepared by the following steps: in a first step, (3-aminopropyl)triethoxysilane or (3-aminopropyl)trimethoxysilane is stirred with water, in a second step, HAuCl.sub.4 is added followed by addition of NaBH.sub.4, to form particles, preferably HAuCl.sub.4 and NaBH.sub.4 are added in the form of solution(s), in a third step, the formed particles are stabilized by bovine serum albumine.

9. The process according to claim 1 for in vitro detection of biomolecules in biological samples, wherein the detection includes interaction of modified particles with said biomolecules, wherein the biomolecules are selected from the group consisting of nucleic acids, proteins, polysaccharides and glycoproteins.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0070] FIGS. 1A-1C: Extinction spectra of modified particles FIG. 1A) A6, A7 and A8, FIG. 1B) B6 and B7, and FIG. 1C) C4, normalized to the absorption maximum. Individual surface modifications do not affect the position of the spectral maxima of series A and series B.

[0071] FIGS. 2A-2B: Dynamic light scattering for modified particles FIG. 2A) A6, A7 and A8, FIG. 2B) B5, B6 and B7 measured in water, PBS buffer (PBS) and 10x concentrated PBS buffer (10x PBS). For both series of particles A and B, a high colloidal stability is evident, characterized by a stable position of the distribution histogram, which is independent of the environment. Exposure of the modified particles to 10x PBS is a robust assay that corresponds to an ionic strength approximately an order of magnitude higher than that present in a physiological environment.

[0072] FIG. 3: Micrographs of modified particles A1, A3, A4, B1, B3, B4 and C4 from a transmission electron microscope. For each of the series A and B, the gradual growth of the particle is demonstrated: A1 and B1 are the starting silica particles, A3 and B3 are silica particles with bound gold nanoparticles, A4 and B4 are compact modified particles. Scale sizes: for A1, A3, A4 particles, the scale is always 100 nm. For particle B1 200 nm, for B3 100 nm, for B4 and C4 200 nm.

[0073] FIG. 4: Detection of human papillomavirus (HPV16) in cervical carcinoma (SiHa) cells by in situ hybridization of viral DNA with detection by modified particles A8. The specific HPV16 signal is visualized by light field microscopy as dark, high contrast spots on a light background located in the nucleus region. In the practical embodiment, specific colors are used: the nucleus is colored with nuclear red (pink color of the nucleus), visualization with A8 provides a dark blue color. Magnification 20x.

[0074] FIGS. 5A and 5B: Analysis of phagocytosis by J774A.1 murine macrophages by flow cytometry without fluorescent labeling. FIG. 5A) Distribution of cells after 3 hours in a control group without modified particles (Control), with modified particles (B6) and with modified particles derivatized with mannose (B7). The process of phagocytosis was monitored using,,dot plots” visualizing the size (direct light scattering, FSC-A) and granularity (lateral light scattering, SSC-A) of individual cells. FIG. 5B) Dynamics of cell distribution in individual quadrants. The specific uptake of modified particles by macrophages is manifested by a significant increase of the signal in quadrant Q1, which also increases with time.

[0075] FIG. 6: Light field microscopy of live macrophages J774A.1 for times 0 to 12 hours. The following variants were tested: medium without particles (Control), medium containing control modified particles B6 carrying only polymer (negative control) and modified particles B7 derivatized with mannose (both particles at a final concentration of 7.3 pmol.1.sup.-1). Phagocytosis was clearly observable only for the specific variant containing B7 with bound mannose as a significant change in optical contrast in the cell areas.

[0076] FIG. 7: Light field microscopy of PBMC cells were isolated from whole blood after 48 h incubation at 4° C. The following variants were tested: medium without particles (Control), medium containing control modified particles C8 carrying only polymer (negative control), modified particles C9 derivatized with CD3 biotinylated monoclonal antibody, and modified particles C10 derivatized with CD4 biotinylated monoclonal antibody. The individual cells containing the corresponding receptors (either CD3 or CD4) show a significant change in optical contrast in the cell areas manifested as dark spots. Notably, not all cells are stained, because PBMC cells contain only a certain fraction of either CD3 and/or CD4 positive cells.

[0077] FIG. 8 schematically shows the structure of a surface-modified particle comprising a core 1. an inner shell 2 and an outer shell 3, where the core 1 is made of silica or it is hollow and has a diameter d.sub.t, the inner shell 2 is formed by a metal layer M of thickness d.sub.2, and the outer shell 3 of thickness d.sub.3 is formed by a layer of polymer.

TABLE-US-00001 List of Abbreviations DCM dichloromethane DIPEA N,N-diisopropylethylamine UPLC-MS ultra high performance liquid chromatography-mass spectrometry TLC thin layer chromatography MeOH methanol DMSO dimethylsulfoxide HRMS high resolution mass spectrometry UPLC ultra-high performance liquid chromatography QDa quadrupole detector in mass spectrometry ACN acetonitrile PEG polyethyleneglycol BTTAA 2-(4-((bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl) acetic acid V-601 dimethyl 2,2′-azobis(2-methylpropionate) PBMC peripheral blood mononuclear cells

EXAMPLES

Example 1

Synthesis of Ligand for Polymerization

[0078] 500 mg of Boc-PEG.sub.4-NH.sub.2 (1.0 eq.) was dissolved in 50 ml of DCM and 446 .Math.l (1.5 eq.) of DIPEA was added to the reaction mixture, which was subsequently cooled in an ice bath. 167 .Math.l (1.0 eq.) of methacryloyl chloride was added dropwise over about 5 minutes. The reaction was allowed to gradually warm to room temperature and stirred overnight. In the morning, the reaction mixture was shaken twice with 10% (always volume percentage, unless otherwise stated) KHSO.sub.4, twice with saturated sodium bicarbonate solution and once with brine, 30 ml each time. The organic phase was then dried over anhydrous magnesium sulphate and evaporated on an evaporator. According to UPLC-MS analysis of the crude product, the residue had a purity of more than 95 % (Rt = 3.7 min, identified m/z: 361.4 [M+H].sup.+, 261.3 [M-Boc, +H].sup.+. TLC analysis (pure EtOAc, R.sub.f = 0.3) confirmed, after staining with 3% KMnO.sub.4, the only major substance present. Product VI was used in the next step without any further purification.

##STR00019##

[0079] The crude evaporated product VI was dissolved in 2 ml of trifluoroacetic acid (TFA) and the solution was sonicated in a bath for 5 min to promote deprotection of the Boc protecting group. The TFA was then evaporated in a stream of nitrogen. After complete removal of TFA, the resulting product VII was used in the next step without further purification.

##STR00020##

639 mg (1.0 eq) of compound VII was dissolved in 50 ml of DCM and then 1.06 ml (3.5 eq) of DIPEA was added. The pH of the reaction mixture was checked to be basic (if the TFA was not removed sufficiently, more DIPEA was needed). 528 mg (1.0 eq) of N-hydroxysuccinimidyl lipoic acid ester was added in one portion and the reaction was stirred overnight. Chromatography on preparative TLC plates (Analtech P02015 Silica GF UNIPLATE, 2000 .Math.m, 200 mm wide, 200 mm length, 4 plates used for separation) was performed as the only purification method (5% MeOH in DCM, R.sub.f = 0.5, UV active). The spot containing the product was scraped off and the product was extracted on a frit with pure MeOH. The substance was further used in the form of a solution in MeOH, because evaporation and concentration of the solution led to spontaneous polymerization. Only a small portion of the solution was evaporated to determine the concentration. 390 mg of product VIII was isolated in MeOH solution (final isolated yield after 3 steps was 51 %). This solution was stable and stored for many months in a freezer at -80° C. UPLC-MS analysis detected only one peak (R.sub.1 = 3.7 min, purity 97 %).

##STR00021##

[0080] For NMR analysis, the solution was gradually evaporated into DMSO. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.92 (NH, t, J = 5.7 Hz, 1H), 7.84 (NH, t, J = 5.6 Hz, 1H), 5.65 (CH.sub.2=, dq, J = 1.9, 1.0 Hz, 1H), 5.32 (CH.sub.2=, h, J = 1.5 Hz, 1H), 4.14 (q, J = 5.2 Hz, 3H), 3.66 - 3.56 (m, 1H), 3.54 - 3.47 (m, 4H), 3.46-3.39 (m, 1H) 3.26 (q, J = 6.1 Hz, 2H), 3.21 - 3.15 (m, 6H), 3.14-3.06 (m, 2H), 2.41 (dtd, J = 12.9, 6.5, 5.5 Hz, 1H), 2.07 (t, J = 7.3 Hz, 2H), 1.93 - 1.79 (m, 4H), 1.66 (dtd, J = 13.6, 7.9, 7.4, 5.5 Hz, 1H), 1.58 - 1.42 (m, 3H), 1.40 - 1.28 (m, 2H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 172.63, 168.06, 140.30, 119.57, 70.18, 70.03, 69.60, 69.26, 56.62, 38.92, 38.55, 35.57, 34.56, 28.74, 25.48, 19.06. HRMS calculated for C.sub.20H.sub.37O.sub.5N.sub.2S.sub.2: m/z [M+H].sup.+: 449.21384; found 449.21378.

[0081] UPLC-MS analysis: data were obtained on a Waters acquity UPLC system connected to a qDA detector. A 2.1×100 mm C18 1.7 .Math.m Acquity Waters was used as the column. The 7-minute gradient was set as follows: 2% ACN for 1 minute, then gradient up to 100% ACN over the next 5 minutes, followed by 1 minute of pure ACN.

Example 2

Preparation of Modified Particles With Silica Core

A) Preparation of Silica Particles

[0082] Silica particles were prepared in two steps. First, particle seed grains were prepared. 4.55 mg of arginine was dissolved in 3.45 ml of water. The solution was overlaid with a mixture of cyclohexane (225 .Math.l) and tetraethoxyorthosilicate (275 .Math.l). The mixture was heated to 60° C. and stirred for 20 hours. Then, the lower phase containing the silica seed grains was separated.

[0083] For further growth of silica particles of various sizes, 39.5 ml of ethanol, 11 ml of H.sub.2O and 466 .Math.l of the prepared solution of silica seed grains were mixed. After thorough mixing, tetraethoxyorthosilicate and 0.9 ml of 24.5% NH.sub.4OH solution were added with stirring. The amount of tetraethoxyorthosilicate was varied according to the desired particle size. For example, to prepare A1 particles with an average core diameter d.sub.1= 58 nm, 180 .Math.l was added; 1000 .Math.l was added to prepare larger B1 particles with an average core diameter d.sub.1 = 95 nm. The reaction was run overnight at room temperature. The particles were washed with ethanol.

B) Preparation of Thiolated Silica Particles

[0084] Particles A1 or B1 (10 mg) prepared in the previous step were mixed with 11.5 ml of water, 37.5 ml of ethanol and the solution was purged with argon for 10 minutes. With stirring, solutions of (3-mercaptopropyl)trimethoxysilane and 1,2-bis(triethoxysilyl)ethane were added in amounts suitable for the given silica particle sizes (to form A2: 14.5 .Math.l to A1 particles; to form B2: 8 .Math.l to particles B1). While stirring, 0.9 ml of 24.5% NH.sub.4OH solution was added and the reaction mixture was allowed to react for 4 hours at room temperature under an inert atmosphere of argon. The prepared thiolated particles were washed with ethanol.

C) Preparation of Silica Particles With Bound Gold Nanoparticles

[0085] First, gold nanoparticles with a diameter of <5 nm were prepared by adding 2 ml of a 1% solution of terachloroauric acid to a solution of tetrakis(hydroxymethyl)phosphonium chloride (1.34 .Math.mol.1.sup.-1 in 50 ml of 10 mmol.1.sup.-1 NaOH solution). The thiol-modified particles (A2, B2) were mixed with this solution. The resulting particles with bound gold nanoparticles (A3, B3) were washed with water.

D) Growth of the Inner Shell

[0086] Particles A3 or B3 were mixed with a gilding solution (250 mg K.sub.2CO.sub.3, 174 mg HAuCl.sub.4, 1 litre water, pH 9.5) and bubbled with carbon monoxide. The ratio of the amount of particles and the gilding solution depended on the particle size and the required thickness of the gold layer. For 0.5 mg of A3 particles, 72 ml of solution was used to form A4 particles, for 0.5 mg of B3 particles, 40 ml of gilding solution was used to form B4. The particles were separated by sedimentation. The average diameter of the inner shell was d.sub.2 = 14 nm for A4 particles and d.sub.2 = 17.5 nm for B4 particles.

Example 3

Preparation of Modified Particles With Hollow Core

[0087] The gold nanoshells with hollow core were prepared according to modified procedure of Guan, Y. et al. (Colloid Surface A, 2016, 502, 6-12). (3-aminopropyl)triethoxysilane (2.0 ml) was mixed with milliQ water (920 ml) under constant stirring. After 10 s, a solution of HAuCl.sub.4 (20 ml, 25 mmol.1.sup.-1) was added to the mixture to form yellow suspension. The reaction mixture was stirred for 30 s, followed by addition of freshly prepared NaBH.sub.4 solution (80 ml, 0.1 mol.1.sup.-1). After addition of NaBH.sub.4, the suspension changed color from yellow to blue. The formed particles with hollow core of average diameter d.sub.1 = 68 nm and diameter of the inner shell d.sub.2 = 12 nm were stabilized by addition of solution of bovine serum albumine (80 ml, 0.1 mmol.1.sup.-1).

Example 4

Preparation of Particles With a Layer of Hydrophilic Polymer

[0088] A mixture of ligand VIII and lipoic acid (14.4 ml of 10 mmol.1.sup.-1 lipoic acid and 3.6 ml of 10 mmol.l.sup.-1 ligand VIII) was added to the gold-plated particles A4 diluted in 700 ml of water, the pH was adjusted to 9.5. Ligand exchange lasted 3 days. The resulting A5 particles were concentrated by sedimentation.

[0089] A mixture of ligand VIII and lipoic acid (9.64 ml of 10 mmol.1.sup.-1 lipoic acid and 2.41 ml of 10 mmol.1.sup.-1 ligand) was added to the B4 particles diluted to 500 ml, the pH was adjusted to 9.5. Ligand exchange lasted 3 days. The resulting B5 particles were concentrated by sedimentation.

[0090] A mixture of ligand VIII and lipoic acid (9.64 ml of 10 mmol.1.sup.-1 lipoic acid and 2.41 ml of 10 mmol.1.sup.-1 ligand) was added to the C4 particles diluted to 500 ml, the pH was adjusted to 9.5. Ligand exchange lasted 3 days. The resulting C5 particles were concentrated by sedimentation.

[0091] A concentrated solution of A5 particles (45 ml corresponding to 5 mg of silica particles) was mixed with a solution of HPMA (9.72 g in 84 ml of water, pH 11) with constant stirring and purged with argon. A mixture of monomer and initiator in methanol was added to the solution under argon [486 .Math.l alkyl of alkyne monomer N-(prop-2-yn-1-yl)methacrylamide, 466 .Math.l of initiator V-601 (Fujifilm, cat. Nr. LB-V601-20GS) in 14.6 ml of methanol]. The mixture was heated to 60° C. for 48 hours. The resulting A6 particles were washed with methanol and then PBS with 0.1% (w/w) Tween detergent.

[0092] A concentrated solution of B5 particles (30 ml corresponding to 6.75 mg of silica particles) was mixed with a solution of HPMA (5 g in 53.75 ml of water, pH 11) with constant stirring and purged with argon. A mixture of monomer and initiator in methanol [250 .Math.l of N-(prop-2-yn-1-yl)methacrylamide, 300 .Math.l of initiator V-601 in 9.375 ml of methanol] was added to the solution under an inert atmosphere of argon. The mixture was heated to 60° C. for 48 hours. The resulting B6 particles were washed with methanol and then PBS with 0.1% (w/w) Tween detergent.

[0093] A concentrated solution of C5 particles (30 ml corresponding to 6.75 mg of silica particles) was mixed with a solution of HPMA (5 g in 53.75 ml of water, pH 11) with constant stirring and purged with argon. A mixture of monomer and initiator in methanol [250 .Math.l of N-(prop-2-yn-1-yl)methacrylamide, 300 .Math.l of initiator V-601 in 9.375 ml of methanol] was added to the solution under an inert atmosphere of argon. The mixture was heated to 60° C. for 48 hours. The resulting C6 particles were washed with methanol and then PBS with 0.1% (w/w) Tween detergent.

[0094] The thickness of the polymer outer shell for the particles A6, B6, and C6 was approximately d.sub.3 = 15 nm.

Example 5

Derivatization of Modified Particles Using Biotin and Neutravidin

[0095] A6 particles weighing 180 .Math.g of silica core were modified with biotin by copper ion catalyzed azide-alkyne cycloaddition. The particles were diluted to 600 .Math.l and mixed with 1 .Math.l 12.5 mmol.1.sup.-1 biotin-PEG.sub.11-azide (BroadPharm, USA, cat. Nr. BP-21626, CAS 956494-20-5). In a final volume of 700 .Math.l, reagent concentrations were used: 77 .Math.mol.1.sup.-1 CuSO.sub.4, 155 .Math.mol.1.sup.-1 BTTAA ligand (Sigma-Aldrich, cat Nr. 906328, CAS 1334179-85-9), 3.86 mmol.1.sup.-1 sodium ascorbate and 3.86 mmol.l.sup.-1 aminoguanidine. The reaction was allowed to react for 2 h and then the A7 particles were washed 7 times in PBS. Half of the A7 particles were mixed with 200 .Math.l of neutravidin in PBS (1 mg/ml) and allowed to react at 4° C. overnight. Particles A8 were washed in PBS with 0.1% (w/w) Tween detergent.

Example 6

Derivatization of Modified Particles Using Mannose

[0096] B6 particles were modified with mannose by copper ion catalyzed azide-alkyne cycloaddition. The particles were diluted to 4.5 ml and mixed with 377 .Math.l of 10 mmol.1.sup.-1 mannose-PEG.sub.4-azide (BroadPharm, USA, cat. Nr. BP-23832, CAS 1632372-86-1) (0.74 mmol.1.sup.-1). In the final volume of 5089 .Math.l, there was 0.15 mmol.1.sup.-1 CuSO.sub.4, 0.3 mmol.1.sup.-1 BTTAA ligand, 1.48 mmol.1.sup.-1 ascorbate and 1.48 mmol.1.sup.-1 aminoguanidine. The reaction was allowed to react for 2 hours and then the mannosylated B7 particles were washed 7 times with PBS with 0.1% (w/w) Tween detergent.

Example 7

Derivatization of Modified Particles Using Antibodies

[0097] C6 particles were modified with biotin by copper ion catalyzed azide-alkyne cycloaddition. The particles were diluted to 600 .Math.l and mixed with 1 .Math.l 12.5 mmol.1.sup.-1 biotin-PEG.sub.11-azide. In a final volume of 700 .Math.l, reagent concentrations were used: 77 .Math.mol.1.sup.-1 CuSO.sub.4, 155 .Math.mol.1.sup.-1 BTTAA ligand, 3.86 mmol.1.sup.-1 sodium ascorbate and 3.86 mmol.1.sup.-1 aminoguanidine. The reaction was allowed to react for 2 hours and then the biotinylated C7 particles were washed 7 times in PBS. Half of the C7 particles were mixed with 200 .Math.l of neutravidin in PBS (1 mg/ml) and allowed to react at 4° C. overnight. The resulting neutravidin-modified particles C8 were washed in PBS with 0.1% (w/w) Tween detergent.

[0098] Particles C8 were incubated with biotinylated immunoglobulin G - CD3 monoclonal antibody (OKT3) (ThermoFischer, cat. Nr. 13-0037-82) for 1 h at room temperature. Afterwards, the resulting particles were washed with 1x PBS with 0.1% Tween 20 and centrifuged at 1000 rcf for 10 min. The supernatant was removed and again centrifuged at 2000 rcf for 10 min, supernatant was discarded. The resuspended pellets were pooled together and the procedure was repeated three times providing particles C9 modified with CD3 monoclonal antibody.

[0099] Another portion of particles C8 was incubated with biotinylated immunoglobulin G - CD4 monoclonal antibody (OKT4) (ThermoFischer, cat. Nr. 13-0048-82) and purified at the same conditions as used above for preparation of C9. The procedure yielded particles C10 modified with CD4 monoclonal antibody.

Example 8

Characterization of Modified Particles

[0100] The extinction spectrum of the prepared modified particles was measured (FIG. 1). At the low picomolar concentration used, all particles showed intense absorption bands in the region suitable for signal detection.

[0101] The dynamic light scattering of the prepared modified particles was measured at concentrations corresponding to units of pmol.l.sup.-1 in water, in PBS buffer and 10x concentrated PBS buffer, which due to its high ionic strength represents a very robust test of colloidal stability (FIG. 2). All modified particles showed high stability even in the environment of 10x concentrated PBS buffer, which indicates a strong stabilizing function of the polymer protecting the interaction interface of the modified particles.

[0102] The structure of the particles in the individual preparation steps was documented by transmission electron microscopy (JEOL JEM-1011, voltage 80 kV) (FIG. 3). The observed changes correspond to the growth mechanism and document the presence of the expected structures, including the resulting “nanoshells”.

Example 9

In Situ Hybridization of Viral DNA in Tumour Cells

[0103] Neutravidinine-derivatized polymer-modified particles (A8) with high optical contrast in transmitted light were used to detect target DNA sequences in the tumour cells genome. In this case, they were tumour cells of cervical carcinoma (SiHa), which are characterized by the presence of 2-3 copies of the integrated form of the HPV16 virus per nucleus. The fixed cells or directly the tumor tissue were immobilized on a galss slide, the DNA was denatured and hybridized with a gene-specific DNA probe labeled with digoxigenin. Excess probe was washed away, and the cells were further incubated with rabbit anti-digoxigenin antibody followed by biotinylated anti-rabbit immunoglobulin antibody. The cells were then incubated with a solution of neutravidin-derivatized A8 particles that bind to biotin. The excess particles were washed away and the nuclei were stained with neutral red for better structural recognition of the cells. The specific HPV16 signal was visualized by light field microscopy as dark, high contrast spots on a light background located in the cell nucleus region (FIG. 4). This example demonstrates the use of modified particles for in vitro diagnostics.

Example 10

Use of Modified Particles for Detection of Phagocytic Activity

[0104] Measurement of the phagocytic activity of professionally phagocytic cells is an indicator of the parameters of non-specific cellular immunity. Its examination is routinely performed clinically from the patient’s blood cells by phagocytosis of fluorescently labeled mannosylated particles (e.g. latex, zymosan, etc.), staining microscopically, or on a flow cytometer. However, fluorescence flow cytometry is an expensive and sophisticated device that is not commonly available in every haematology laboratory. Due to the extreme scattering properties of the modified particles, they can be used to measure phagocytic activity, e.g. by flow cytometry in native, fluorescently unstained cells, by haemocytometry, or by light field microscopy with automated image analysis. The murine cell line J774A.1 (ATCC® TIB-67™) cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum and antibiotics (100 U/100 .Math.g penicillin /streptomycin) was used as model macrophages.

Cytometric Detection

[0105] The day before the experiment, 6×10.sup.5 J774A.1 cells were seeded on a 60 mm culture dish. After 24 hours, fresh medium without particles (control), medium containing control modified particles B6 carrying only polymer (negative control) and modified particles B7 with bound mannose (both particles at a final concentration of 7.3 pmol.1.sup.-1) were added. Cells were harvested at 0, 1, 3 and 6 h intervals after the addition of medium with or without particles and subsequently measured without fixation by light scattering detection. The results are summarized in FIG. 5. It can be seen that the mannosylated particles B7 were selectively and intensively taken up by macrophages, the uptake was time-dependent, and that the cytometric method without the use of fluorescent labeling was very sensitive for the detection of phagocytic activity.

Detection by Light Field Microscopy

[0106] The day before the experiment, 1×10.sup.4 J774A.1 cells were seeded in a 96-well plate. After 24 hours, fresh medium without particles (control), medium containing control modified particles B6 carrying only polymer (negative control) and modified particles B7 with bound mannose (both particles at a final concentration of 7.3 pmol.1.sup.-1) were added. Live cell imaging at 0, 1, 3, 6, 12, and 18 h after addition of medium with or without particles was performed using a high-capacity analysis in a microscope discovery system Cell Voyager CV7000 (Yokogawa, Japan) at 37° C. in 5% CO.sub.2 in light field mode. The results are summarized in FIG. 6. As in the case of cytometric detection, the microscopic method without the use of fluorescent labeling was very sensitive for the detection of phagocytic activity. Mannosylated particles B7 were selectively and intensively taken up by macrophages and uptake was time-dependent.

Example 11

Detection of CD3+ and CD4+ T Lymphocytes

[0107] This Example demonstrates the use of particles C9 and C10 modified with monoclonal antibodies CD3 or CD4, respectively, as a staining tool for CD3+ or CD4+ T lymphocytes, respectively. The number of CD4+ T lymphocytes in blood is supressed in the patients who suffer form Human Immunodeficiency Virus (HIV). Counting CD4+ T lymphocytes is one of the most useful parameters for following the progress of HIV disease.

[0108] PBMC cells were isolated from whole blood using density gradient centrifugation (Ficoll™), seeded in CellCarrier-96 well plate (PerkinElmer) at 5 × 10.sup.4 cells/well and incubated with C9 particles modified with CD3 monoclonal antibody or with C10 particles modified with CD4 monoclonal antibody for 48 h at 4° C. The cells were imaged using PerkinElmer Operetta imaging system. Bright field images were captured by 40x long WD objective. All images were post-processed using integrated Operetta system Harmony 4.1 and ImageJ software. The results are summarized in FIG. 7. The microscopic method without the use of fluorescent labeling was very sensitive for the detection of the specific cells displaying the target receptors. The control particles C8, which did not contain any antibody, did not stained the cells. In contrast, the fraction of cells containing CD3 receptor was stained by particles C9, and the fraction of cells containing CD4 receptor was stained by particles C10 (FIG. 7). After further optimisation and validation this method can be used as a quantitative tool for counting the number of circulating the level in whole blood.

INDUSTRIAL APPLICABILITY

[0109] The present invention solves the problem of direct detection in in vitro diagnostics by light extinction using surface-modified particles with a non-metallic core coated with a metal layer on which a biocompatible polymer brush is attached. Their application allows the direct detection of biomolecules including nucleic acids, proteins, polysaccharides and glycoproteins in biological samples. By biological sample is meant, for example, blood, blood plasma, blood serum, urine, semen, tears, saliva, mucus, stool, sweat, swab, lymph, cerebrospinal fluid, cell suspension or tissue sample.