METHOD OF MAKING NANOPARTICLES IN AN AQUEOUS SOLUTION PROVIDING FUNCTIONALIZATION AND HINDERED AGGREGATION IN ONE STEP

Abstract

The invention relates to a method of making a functionalized nanoparticle in an aqueous solution, wherein a chemical functionalization of a metal nanoparticle in the aqueous solution is provided and the aqueous solution comprises water and ingredients. The ingredients comprise at least the metal nanoparticle, a thiol of the form R—SH, where R represents a substituent, and a silver compound. The invention further relates to a plurality of functionalized nanoparticles according to the method, wherein each of the plurality of functionalized nanoparticles comprises a metal core, a silver coating and a sulfide bond substituent. The invention also relates to a lateral flow test method and device.

Claims

1. A method of preparing a functionalized nanoparticle, comprising a metal core, a silver coating and a sulfide bond substituent, in an aqueous solution, the method comprising a step of chemical functionalization of a metal nanoparticle in the aqueous solution, wherein the aqueous solution comprises water and ingredients, wherein the ingredients comprise the metal nanoparticle, a thiol of the form R—SH, where R represents an organic substituent having a functional group, and a silver compound.

2. The method according to claim 1, wherein silver of the silver compound is deposited on the metal nanoparticle by wet chemical reaction.

3. The method according to claim 1, wherein the ingredients are provided in one step, wherein, in particular, a plurality of the metal nanoparticles is functionalized such that aggregation of the plurality of functionalized nanoparticles is prevented after the wet chemical reaction has finished.

4. The method according to claim 1, wherein the organic substituent comprises an oligonucleotide, a Polyethylene glycol (PEG or mPEG), or MPA.

5. The method according to claim 1, wherein the metal nanoparticles provided comprise nanospheres and/or nanorods.

6. The method according to claim 1, wherein the functional group comprises a carboxyl group, an aldehyde group, a hydroxyl group, an amino group, or an amide group.

7. A plurality of functionalized nanoparticles, wherein each of the plurality of functionalized nanoparticles comprises a metal core, a silver coating and a sulfide bond substituent.

8. The plurality of functionalized nanoparticles according to claim 7, wherein the metal core comprises one or more of the following metals: Au, Ag, Al, Pt, Pd, Cu, Rh, Fe.

9. The plurality of functionalized nanoparticles according to claim 7, wherein the silver coating of each of the functionalized nanoparticles forms a shell around the metal core and the metal core is at least partially covered by the silver shell.

10. The plurality of functionalized nanoparticles according to claim 7, wherein the sulfide bond substituent protrudes from the silver coating.

11. The plurality of functionalized nanoparticles according to claim 7, wherein the sulfide bond substituent exceeds the thickness of the silver coating.

12. A plurality of functionalized nanoparticles, wherein each of the functionalized nanoparticles comprises a metal core, a silver coating and a sulfide bond substituent, and wherein each functionalized nanoparticle is synthesized by a method of preparing a functionalized nanoparticle in an aqueous solution, comprising a step of chemical functionalization of a metal nanoparticle in the aqueous solution, wherein the aqueous solution comprises water and ingredients, which are selected from the group consisting of the metal nanoparticle, a thiol of the form R—SH, where R represents a substituent, and a silver compound.

13. A nanoscale object functionalized with at least one functionalized nanoparticle synthesized by the method according to claim 1.

14. A test device for performing a lateral flow test, which contains a test substrate including a plurality of functionalized nanoparticles according to claim 7.

15. (canceled)

16. The method according to claim 1, wherein the organic substituent comprises one or more of an amino acid, a protein, an antibody, a virus, and a hormone.

17. The method according to claim 1, wherein the silver forms a shell around the metal nanoparticle.

18. The method according to claim 17, wherein the thiol attaches onto the silver of the silver shell by forming a sulfide bond with the silver of the shell.

19. The method according to claim 4, wherein the oligonucleotide is an RNA, a PNA, or a DNA.

20. The method according to claim 4, wherein the oligonucleotide comprises sequences of bases selected from adenine (A), cytosine (C), guanine (G) or thymine (T).

21. The plurality of functionalized nanoparticles according to claim 7, wherein the sulfide bond substituent comprises an oligonucleotide, a polyethylene glycol (PEG or mPEG), or MPA.

Description

[0073] Further advantages, features and applications of the present invention are provided in the following detailed description of the exemplary embodiments and the appended figures. The same components of the exemplary embodiments are substantially characterized by the same reference signs, except if referred to otherwise or if other reference signs emerge from the context. In detail:

[0074] FIG. 1 schematically illustrates the cross sectional view of a spherical and a rod shaped functionalized nanoparticle according to the invention.

[0075] FIG. 2 schematically illustrates an enlarged cross sectional view of the functionalized nanoparticle according to the invention.

[0076] FIG. 3a shows a top and a bottom image. In the top image an electron microscopy recording of two Au/Ag rod shaped DNA functionalized nanoparticles according to the invention is shown. The bottom image shows a corresponding zoom out view of a plurality of Au/Ag DNA functionalized nanorods. The scale bar is 50 nm in each case.

[0077] FIG. 3b shows Au/Ag rod shaped DNA functionalized nanoparticles according to the invention (top and bottom zoom out image). The scale bar is 50 nm in each case. The density of the DNA substituents was further increased compared to FIG. 3a. The DNA functionalization layer then appears as a more prominent white layer around the Au/Ag nanorods (sample stained with Uranyl Formate).

[0078] FIG. 3c shows one (top) and two (bottom) of the Au/Ag rod shaped DNA functionalized nanoparticles of FIG. 3b attached to a DNA origami template. The scale bas is 50 nm.

[0079] FIG. 4 schematically represents the individual steps 101-104 of preparing the aqueous solution and of starting the wet chemical reaction.

[0080] FIG. 5 schematically illustrates a particular embodiment of making the functionalized nanoparticle according to the invention.

[0081] FIG. 6 schematically illustrates an embodiment of the application of the functionalized nanoparticles at a DNA origami pattern.

[0082] FIG. 7a shows an embodiment of a test device for performing a lateral flow test according to the invention, in a first status of its application.

[0083] FIG. 7b shows the test device of FIG. 7a, in a second status of its application.

[0084] FIG. 8 shows a diagram describing the method of producing a test device for performing a lateral flow test according to the invention.

[0085] FIG. 1 shows a cross sectional view of the functionalized spherical nanoparticle 1 and a functionalized nanorod-shaped nanoparticle 1′ according to an embodiment of the invention having, respectively a spherical or nanorod-shaped metal core 2, 2′, e.g. a core made of Au, marked with dots and a silver coating 3, 3′ shown dashed. The silver coating forms a shell 3, 3′ surrounding the metal core 2, 2′. The silver coating is thin compared to the dimensions of the metal core. The thickness of the silver shell 3, 3′ can be tuned. A lower concentration of, for example Au nanorods 2′ and or a higher concentration of for example, AgNO3 will result in a thicker shell 3′. The silver shell 3, 3′ has several substituents 5, 5′ or ligands are attached to it, as indicated in FIG. 1. The number of substituents 5, 5′ attached to the silver surface may vary. The number of substituents shown in the FIG. 1 does not represent their actual surface concentration. The number of substituents 5, 5′ attached to the silver shell can, for example, be adapted by changing the number of modified substituents (R—SH) in the aqueous solution. The substituents 5, 5′ can form a further layer 4, 4′ surrounding the silver shell 3, 3′. The thickness of the substituent layer 4, 4′ or ligand layer is variable, e.g. depends on the substituent 5, 5′ dimensions, e.g. molecular length or folded structure, e.g. coil shape, when attached. In a most preferred embodiment, a DNA substituent 5, 5′ comprises 19 thymine nucleotides (T19). The further layer 4, 4′ comprising the substituents 5, 5′ also serves to increase stability of the functionalized nanoparticles 1, 1′ in solution, i.e. it acts as a stabilizing layer. In particular when DNA molecules are used as substituents 5, 5′ the solution of functionalized nanoparticles is stable even upon freezing and thawing or upon centrifugation and including re-dilution of the centrifuged particles 1, 1′. The aqueous solution was based on ddH2O. The aqueous solution did not contain bivalent cations, e.g. Ca2+, Mg2+, which is generally preferred for generating the functionalized nanoparticle according to the invention. Preferably, the aqueous solution, in particular for storing the functionalized nanoparticles, contains salt, in particular bivalent cations, in a concentration, each preferably, up to 1 mM, 2 mM, 3 mM, 4 mM, 5 mM.

[0086] FIG. 2 shows an enlarged schematic cross sectional view of the functionalized nanoparticle 1, 1′ according to an embodiment of the invention. In the embodiment of FIG. 2, the Au core 2, 2′ of FIG. 1 is indicated by dots as a bottom layer. On top of this layer, the silver shell 3, 3′ of FIG. 1 is formed as indicated by a plurality of circles 6. Each circle 6 indicates an individual silver atom 6 of the shell 3, 3′. The silver atoms 6 are drawn as horizontally neighbored and as packed in a vertical direction. Thereby, the silver layer is formed in the vertical direction by three densely packed layers of horizontally neighbored silver atoms 6. The top silver atom layer relates to the surface of the shell 3, 3′ and is exposed to the aqueous solution. Further, one silver atom 6 of the top silver layer has a sulfide bond formed to it through the sulfur atom 7. The sulfur atom 7 is further covalently bond to the substituent 5, 5′, which in the embodiment shown is a DNA strand 5, 5′. In a preferred embodiment, the tiol is a HS-T19 modified DNA strand. The deposition of silver happens preferentially in certain facets. The geometry due to faceting becomes more apparent, the thicker the Ag shell is. That is the geometry of the grown silver shell deviates more and more from the original geometry of the metal core. For example, the shape of the silver shell initially appears as the original rod shape until it turns towards, e.g. a rhombic shape, when the silver layer is significantly grown.

[0087] Further indicated in FIG. 2 is the size of the attached HS-T19 DNA strand, marked as “dDNA”. Also the vertical height of the densely packed silver atom layers 3, 3′ is indicated d.sub.Ag. By means of X-ray scattering structural details of the Au/Ag core-shell nanorods in solution can be accessed. In the particular embodiment of FIG. 2 small angle (SAXS) and wide angle (WAXS) X-ray scattering was performed. The SAXS intensities for two geometries are then model fitted considering a cylindrical core-shell-shell particle geometry. From the SAXS data a radius of the Au core of r.sub.AU=34 Å with a polydispersity (PD) ratio of 0.1, a thickness of the Ag shell with d.sub.Ag=5 Å, and a thickness of the DNA shell with d.sub.DNA=29 Å is obtained. Also the length of the Au nanorod core of L.sub.AU=155 Å with a PD ratio of 0.3 is obtained. The measured parameters can additionally be compared to the dimensions obtained from transmission electron microscopy (TEM) imaging. Moreover, the SAXS data additionally serve to indicate a closed cover of the Au core 2, 2′ by the silver shell 3. That is the silver shell 3, 3′ forms a continuous layer on the metal core 2, 2′. The Au core 2, 2′ is homogeneously covered by the shell 3, 3′. The SAXS data thus can be used to verify the exclusion of porosity of the Ag shell. The X-ray data further serve to proof binding of the substituent onto the shell.

[0088] Further verification of the crystallinity and crystal structure of the Au/Ag core-shell nanorods, and the Au nanorods is obtained by comparison of WAXS data, having the nanorods functionalized with and without DNA substituent 5, 5′. From the WAXS profiles fcc diffraction peaks with a refined lattice parameter for the Au nanorods, a lattice parameter for the Au/Ag nanorods without DNA shell, and a lattice parameter for the Au/Ag nanorods with DNA shell are obtained.

[0089] FIG. 3a shows two electron microscopy images in a bottom and a top zoomed view. The images represent the DNA— stabilized Au/Ag core shell nanorods according to one preferred embodiment of the invention. The scale bar is 50 nm. The DNA layer 4′ formed on the surface of the silver shell 3′ of the functionalized nanorods appears as a thin white layer in the top zoom image.

[0090] Accordingly, FIG. 3b shows two further electron microscopy images representing a further embodiment of the DNA— stabilized Au/Ag core shell nanorods, wherein the number of attached DNA substituents 5′ on the shell 3′, forming the respective DNA layer 4′ is increased. The feature can be recognized as the white layer 4′ appears brighter. A denser DNA loading is achieved by freezing and thawing the solution containing the functionalized nanoparticles.

[0091] Whether the wet chemical reaction is finished appears from optically inspecting the solution after the reaction has started. That is upon silver growth and functionalization a color change of the solution visibly appears. The synthesized DNA— stabilized Au/Ag core shell nanorods are then frozen. The presence of DNA on the particle's surface and in solution prevents the functionalized nanoparticles from aggregation upon freezing. The freezing procedure gives rise to an increased DNA loading owing to the excess DNA. It is noteworthy, that after a removal of the excess DNA the Au/Ag nanorods comprising DNA can be frozen as well, which further demonstrates their stability. In contrast, conventionally stabilized nanoparticles aggregate immediately and irreversibly upon freezing. A further advantage provided here is the possibility of a long-term storage of the Au/Ag nanorods comprising DNA in the frozen state, which makes them equally convenient for use as the Au nanoparticles. Further, neither a change in quality, i.e. stability, nor in their optical properties takes place. Neither, after different freezing durations or freezing and thawing cycles.

[0092] In order to determine the number of DNA loaded onto the silver shell and using the method according to the invention, a displacement reaction using dithiothreitol (DTT) can be performed. Upon addition of DTT to the solution comprising the Au/Ag nanorods with DNA attached, the conjugated DNA is released as the DTT exhibits a higher affinity to the metal surface. The Au/Ag nanorods comprising DTT are then removed from the solution by centrifugation and the DNA concentration in the solution can be determined by UV/vis spectroscopy, which then can be related to the concentration of nanorods. Alternatively, fluorescently labeled DNA strands can be used as to-be-displaced molecule to increase the sensitivity.

[0093] FIG. 3c shows two electron microscopy images of an embodiment of a functionalized nanorod 1′. In the top image, the DNA functionalized nanorod 1′ is attached to a DNA origami template 8. In the bottom image, two DNA functionalized nanorods 1′ are attached to the origami template 8, wherein the origami template 8 is aligned between the two particles 1′ and along their longitudinal direction. In the particular embodiment shown in FIG. 3c, attachment of the particle 1′ to the actual origami structures 8 occurs via binding of the functional substituent 5′ to both, the silver shell 3′ and the origami structure 8. The origami structure 8 can be any nano structure or nano sized object and the particles 1′ can be either spherical or non-spherical particles prepared according to the method provided by the invention.

[0094] FIG. 4 illustrates an embodiment of the individual process steps according to the method of the invention. In a first process step 101 of the claimed method metal nanoparticles 2′, for example Au nanorods, which form the core nanoparticles 2′ are re-dispersed in a CTAB solution. In a second step 102 the thiol-ligand is added in an excess amount along with AgNO.sub.3 and a reducing agent, e.g. L-ascorbic acid, to the as-prepared nanoparticles 2′, e.g. the Au nanorods. In a third step 103 the pH is raised by adding NaOH which initiates the redox reaction. In a fourth step 104, during Ag-shell 3′ growth, the ligand 5′ binds to the Ag-shell 3′ imparting instantaneous stabilization and functionalization. Hence functionalization and stabilization of the grown silver coated metal core nanoparticles 1′ is provided in one step.

[0095] The Ag-shell 3′ is grown in the presence of a functional ligand, for example DNA-SH, MPA or mPEG-SH, which allows for their immediate conjugation without having the steric interference of a stabilizer. The stability provided by the ligand 3′ is considerably higher compared to the conventional stabilizers. This can be proven in that the nanoparticles 1′ can be redispersed in different media without having a desorption of the stabilizing layer 4′. A desorption of the stabilizing layer 4′ would result in the aggregation of the nanoparticles 1′. Aggregation can be observed either by bare eye, since the solution becomes optically transparent or means absorption spectroscopy.

[0096] FIG. 5 schematically illustrates an embodiment of the method of making a functionalized nanoparticle according to the invention in detail. All chemical ingredients such as HAuCl.sub.4, AgNO.sub.3, CTAB, NaOH, L-ascorbic acid, MgCl.sub.2, sodium citrate, thiol-DNA, SDS, are used as received.

[0097] Not shown in FIG. 5 is the synthesis of gold nanorods 2. The synthesis of Au nanorods 2 is carried out following known protocols in literature, for example ACS nano, Vol. 6, 2012, pages 2804-2817, X. Ye, L. Jin, H. Caglayan, J. Chen, G. Xing, C. Zheng, V. Doan-Nguyen, Y. Kang, N. Engheta, C. R. Kagan, C. B. Murray.

[0098] Step A: After synthesis of the Au nanorods 2, the Au nanorods 2 were re-dispersed in a solution 9 of 0.1 M CTAB in a beaker 10.

[0099] Step B: 5 mL of the Au nanorods 2 22.5 mL of 0.1 M CTAB and 2.5 mL of 100 μM of thiol-modified DNA 5 are added. CTAB crystallizes at room temperature and therefore the mixture is stirred and heated to 30° C. and is kept under this temperature to ensure the dissolution of CTAB.

[0100] Step C: 4 mL of 2 mM AgNO3 and 625 μL of freshly prepared 0.2 M L-ascorbic acid are added.

[0101] Step D: 1.25 mL of 0.2 M NaOH is added to increase the pH and the reduction potential of L-ascorbic acid. Upon pH increase the wet chemical reaction starts.

[0102] Step E: After a few seconds a color change can be observed. The reaction is completed a few minutes after the color change. The obtained stable Au/Ag core-shell functionalized nanorods 1 are further isolated from the reaction solution by 4-times centrifugation, for example at 5000 rpm (2350 rcf) depending on the particles size for 20 min and re-dispersion in 0.1% SDS (not shown).

[0103] FIG. 6 illustrates an embodiment wherein the functionalized nanoparticles 1, 1′ according to the invention are attached to a nano structure 8. Attaching the nanoparticles 1, 1′ to the nano structure 8 is accomplished through the substituents 5, 5′. Thereby different types of nanoparticles, e.g. nanorods and nanospheres are used, each having respective metal cores 2, 2′ and a silver shell 3, 3′. In the particular embodiment shown in FIG. 5 the nano structure 8 is an origami template, in particular a DNA origami, wherein the functionalized nanoparticles 1, 1′ are attached to form a nano object 11. Several nano objects 11 can be obtained by attaching the nanoparticles 1, 1′ to them, whereas the individual nano objects 11 are distinguishable by different chirality. A solution containing for example the plurality of the produced nano objects 11 is optically active in a way that polarized light passing through the solution will be rotated. Alternatively, the nanoparticles 1, 1′ can be attached onto a surface, in particular attached to a surface according to a predefined pattern, whereas selective adsorption of the nanoparticles 1, 1′ along the predefined pattern occurs through the nanoparticle functionalization. The attached nanoparticles 1, 1′ then serve through their silver metal properties to guide or scatter a light beam towards a certain direction.

[0104] In an another application of the functionalized nanoparticles 1, 1′ fluorophores are further attached to the substituents 5, 5′ and the functionalized nanoparticles 1, 1′ are then used as marker molecules to observe selective binding reactions, in particular binding of medical agents, whereas long time studies are possible, because of the achieved enhanced stability of the functionalized nanoparticles 1, 1′ provided by the invention. Before the actual use of the specifically labeled functionalized nanoparticles 1, 1′, the particles 1, 1′ can be readily synthesized, labeled with fluorophores and stored by freezing without losing their advantageous effects.

[0105] While above at least one exemplary embodiment of the present invention has been described, it has to be noted that a great number of variation thereto exists. Furthermore, it is appreciated that the described exemplary embodiments only illustrate non-limiting examples of how the present invention can be implemented and that it is not intended to limit the scope, the application or the configuration of the herein-described nanoparticles and methods relating thereto. Rather, the preceding description will provide the person skilled in the art with constructions for implementing at least one exemplary embodiment of the invention, wherein it has to be understood that various changes of functionality and the arrangement of the elements of the exemplary embodiment can be made, without delegating from the subject-matter defined by the appended claims and their legal equivalents.

[0106] FIG. 7a shows a test device 200 for performing a lateral flow test according to the invention, in a first status of its application. FIG. 7b shows the test device 200 of FIG. 7a, in a second status of its application. The test device comprises a test strip 201, made from a porous material, e.g. containing cellulose. The porous material has the ability to let a fluid sample 222, for example a medical body liquid, or an aqueous dilution containing the same, flow along a direction F parallel to a length axis of the test strip 201, driven by capillary forces. In a region 202 of the test strip, the functionalized nanoparticles according to the invention (or in case of multiplexing: different groups of different functionalized nanoparticles) are located, acting as visual markers for specifically binding to a target.

[0107] The test device is preferably configured to perform a so-called sandwich assay. Sandwich assays may be generally used for larger analytes because they tend to have multiple binding sites. As the fluid sample 222 migrates through the test strip it first encounters a conjugate, which is an antibody specific to the target analyte labelled with the visual marked, which is a functionalized nanoparticle according to the invention. The antibodies bind to the target analyte within the sample fluid and migrate together until they reach the test line 203. The test line 203 also contains immobilized antibodies specific to the target analyte, which bind to the migrated analyte bound conjugate molecules. The test line then presents a visual change 203′ due to the concentrated visual marker, hence confirming the presence of the target molecules. In case of multiplexing, different groups of different nanoparticles are provided in region 202, and different test lines 203 are located at different positions along the length of the test strip.

[0108] FIG. 8 shows a diagram describing the method of producing a test device for performing a lateral flow test according to the invention, including the steps of providing a test substrate; (301) and applying to the test substrate a plurality of functionalized nanoparticles according to the invention and/or a nanoscale object according to the invention (302).

LIST OF REFERENCE SIGNS

[0109] 1 Functionalized nanoparticle [0110] 2 Metal core [0111] 3 Silver coating [0112] 4 Substituent layer [0113] 5 Substituent [0114] 6 Silver atom [0115] 7 Sulfur atom [0116] 8 Nano structure [0117] 9 Solution [0118] 10 Beaker [0119] 11 Nano object [0120] 200 Test device [0121] 201 Test substrate [0122] 202 Region containing the functionalized nanoparticles plus its mobile conjugate [0123] 203 Test lines with immobilized antibodies for letting the conjugate bind to the antibodies [0124] 300 Method of producing the test device [0125] 301, 302 method steps of method 300