WO2004083377A2

WO2004083377A2 - Microorganism-templated nanoparticle assembly

Info

Publication number: WO2004083377A2
Application number: PCT/US2003/025998
Authority: WO
Inventors: Chad A. Mirkin; Zhi Li; Jwa-Min Nam
Original assignee: Northwestern University
Priority date: 2002-08-16
Filing date: 2003-08-18
Publication date: 2004-09-30
Also published as: AU2003302725A8; WO2004083377A3; AU2003302725A1

Abstract

The present invention provides a method of using microorganisms as templates to massively organize nanometer-sized “bricks” into ordered micro-structures, and eventually to functional macro-materials. The method is used to construct nanoparticles into size and shape controllable micrometer-structures using rod or sphere shaped microorganisms as templates. This bio-templating strategy is applicable to contruct micrometer-structures and materials by using magnetic, semiconductive, and noble nanoparticles with or without the help of biological engineering techniques.

Description

Microorganism-Templated Nanoparticle Assembly

FIELD OF THE INVENTION

The invention resides in the field of nanotechnology and particularly nanoparticle assembly.

BACKGROUND OF THE INVENTION

There has been significant progress made in the discovery and refinement of methods for printing and constructing nanostrucrures composed of a wide range of compounds on a variety of surfaces. Methods such as Dip Pen Nanolithography (DPN), printing, stamping and photolithography are leading to significant advances in the understanding of the chemical and physical consequences of miniaturization and the application of patterned surfaces in fields ranging from microbiology to electronics to catalysis. Despite these recent advances in producing nanostrucrures, the ability to manipulate the nanostrucrures and assemble them into more complex structures has not shown such rapid development. Although enormous efforts have been made to assemble pre-existing nanometer-sized building blocks into multi-dimensional ordered structures, materials, and devices using different approaches, there are only a limited number of methods to efficiently organize nanoscopic objects into well defined microscopic structures, and macroscopic sized functional materials.

It has been suggested that the microscopic dimension and complex architecture of microorganisms offers a template for the assembly of nanostrucrures into structures having microscopic dimensions. Previous research directed to the biomineralization phenomena in microorganisms studied calcification, silica deposition and formation of other biogenic minerals by bacteria, lichen, algae, and fungi. However, these approaches rely on the functional properties of live microorganisms acting on individual molecules or metal atoms to concentrate and assemble nano- or microstructures. Thus, there is still a need for a reliable method of using microorganisms as effective templates with which to order a wide range of micrometer-sized nanoparticle structures. SUMMARY OF THE INVENTION hi this invention, the cell structures of microorganisms are used as "bio-templates" to effectively assemble metal particles into ordered micrometer-sized nanoparticle structures (cylinders, belts and rings). Additionally, by modifying nanometer-sized building blocks with oligonucleotide strands, these structures gain an extra recognition property. The bio-templated synthetic method can be used to build up a wide range of ordered nanoparticle structures forming new materials with special optical, electrical and catalytic properties.

One embodiment of the present invention provides a method of fabricating microstructures in which microorganisms are inoculated into a growth media containing metal nanoparticles. The microorganisms are then grown in the media to accumulate the metal nanoparticles within and on the microorganism. The microorganisms are then treated to form a microstructure of metal nanoparticles templated from the microorganism. The metal nanoparticles may be gold or silver nanoparticles having a diameter between about 5 nm and 100 nm. The microorganism is preferably a rod or sphere-shaped fungal or bacterial microorganism. The microorganisms containing metal nanoparticles are then treated to form the micro or macro-structures having the size and shape characteristics imparted by the microorganim template. The metal nanoparticle may have surface modifications such as thiolated oligonucleotides and Bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt which impart recognition properties to the nanoparticles and therefore to the microstructure.

Another embodiment of the present invention is a rod or sphere shaped microorganism formed by the method described above with a metal nanoparticle having a surface modification accumulated on a surface of the microorganism. The surface modifications can include a thiolated oligonucleotide and/or bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt. The microorganism may be bacterial or fungal. The metal nanoparticles may be gold and/or silver having a diameter between about 5 nm and about 100 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows gold nanoparticles accumulated by the filamentous fungus, Aspergillus niger. (a) 0.5 ^χM9 minimal media impregnated with about 13 nm Gold colloid solution (approximately 7 nM) for fungal growth. The red color is characteristic of the Gold colloid, (b) a reddish purple fungal mycelium formed after inoculation of the media in Figure la with Aspergillus niger, (c) UV-Vis spectra of the media during the mycelium growing process after inoculation of Aspergillus niger, (d) optical microscopic image of the fringe of the mycelium in Figure lc. Figures 2a-c show TEM images of an individual Aspergillus niger hypha loaded with Gold nanoparticles with different magnifications, (d) section image of an Aspergillus niger hypha loaded with Gold nanoparticle embedded in resin (e) higher resolution image of the "Gold nanoparticle necklace" of Figure 2d.

Figure 3 a is a schematic representation of the use of DNA hybridization to assemble a layer of 30 nm Gold particles onto the surface of a 13 nm gold particle cylindrical structure supported by Aspergillus niger hypha, (b and c) transmission electron microscopy images of a thin section of a hypha, subsequently loaded with 13 nm Gold nanoparticles, and then assembled with 30 nm Gold nanoparticles through DNA hybridization. Figure 4a, shows a thin film with golden color formed by air-drying Aspergillus niger mycelium-loaded Gold nanoparticles. (b) FE-SEM image of the surface of the thin film of Figure 4a. Belt shaped nanoparticle structures (approximately 5 to 6 μm in width) supported by fungal hyphae are clearly seen.

Figure 5. The conductivity as a function of temperature for a thin film of Aspergillus niger mycelium loaded with Gold nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of using bio-templates in the controlled assembly of nanoparticles into ordered structures, hi one embodiment of the present invention, fungal mycelium are used as bio-templates to assemble nanoparticles into ordered structures. Mold and actinomycetes are composed of branched hyphae structures which create a percolating network of filamentous fungal mycelium. Micrometer or nanometer-sized metal crystals such as gold and silver can be produced by certain microorganism species through bio-reduction of metal ions or metal complexes. Microorganisms and related biopolymers can accumulate heavy metal ions from their surrounding environments through bio-sorption. i one embodiment of the present invention, a spore of a filamentous fungus is first inoculated into a growth media containing a metal nanoparticle. Any rod or sphere- shaped mold or actinomycetes species forming the mycelium may be used. Preferred species include Penicilliun notatum, Rhizopus stolonifer, Mucor hiemalis, Streptomyces venezuelae and Aspergillus niger. Most preferably, the filamentous fungus Aspergillus niger is used. Additionally, killed fungi and actinomycetes bio-mass can also be used to accumulate the metal nanoparticles from the growth media.

The metal nanoparticles can be any metal atom that can be accumulated on the surface of a microorganism such as gold or silver. The nanoparticles can be produced by a variety of methods known in the art. For example, gold-thiolated nanoparticles are prepared and modified with thiolated oligonucleotide strands by standard methods (Storhoff et al. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes. J. Am. Chem. Soc, 120, 1959-1964 (1998); Li, Z., Jin, R., Mirkin, C. A., Letsinger, R. L. Multiple Thiol-Anchor Capped DNA-Gold Nanoparticle Conjugates. Nucleic Acids Res. 30, 1558-1562 (2002)). The metal particles may range in size between about 5 nm and about 100 nm. Preferably, gold particles are used having a diameter between about 10 nm and about 40 nm. More preferably, gold particles for use in the present invention are prepared with a diameter between about 13 nm and about 30 nm.

The modified metal nanoparticles are dispersed in a suitable growth media for the filamentous microorganism. Any media that will support adequate growth of the microorganism during the assembly of the metal structures is sufficient. A 0.5 xM9 minimal media solution (21 mM Na2HPO4, 11 mM KH₂PO₄, 9.3 mM NH₄C1, 4.3 M NaCl, 0.014 mM CaCl₂, 0.5 mM MgSO₄, 0.01% Glucose) has been used to support the growth of Aspergillus niger with good results. The concentration of the metal nanoparticles in the growth media may vary widely within a range that does not kill or inhibit the growth of the microorganisms. For example, the gold-thiolated nanoparticles described above may be added to the growth media at a concentration of about 7nM with good results.

Following inoculation of the growth media containing the metal nanoparticles, the media is maintained under appropriate conditions to support bacterial growth or spore gennination and continued fungal growth. During this phase of the assembly, the actinomycetes or fungal hyphae continue to grow and branch, while the metal nanoparticles in the media begin to assemble onto the surface of the microorganism resulting in an increase in the size of the bacteria or fungal mycelium. During this phase, the color of media may be monitored by UV-Vis spectroscopy to evaluate uptake of the metal nanoparticles from the media. Typically, the removal of metal nanoparticles from the media and assembly onto the surface of the bacteria or fungal hyphae results in a concomitant decrease in the color of the media as observed by UV-Vis spectroscopy. At a certain point, the media loses most of its color indicating that most of the Gold nanoparticles are assembled into bacteria or mycelium pellets.

Actinomycetes or hypha loaded with nanoparticles are then dried which causes them to contract into a belt shape with nanoparticles that are densely packed along the belt. The microorganisms can be directly air-dried or dehydrated with acetone. As shown in Figure 4a, the dried microorganisms can be formed into a thin film of having a thickness of about 0.2 mm. Field Emission Scaning Electron Microscopy (FE-SEM) images of the surface morphology of this golden thin film with a metallic gloss are shown in Figure 4b. Transmission Electron Microscopy (TEM) images of the individual hypha are shown in Figures 2a - 2c. The electrical conductivities of thin films of different fungal species vary from about 10^" to 10^" Scm^" at room temperature. As shown in Figure 5, the temperature-dependent conductivities of these mycelium films follows Arrhenius behavior due to a thermally activated charge transport between individual Gold nanoparticles. Both this semiconductive behavior and the activation energy (Ea = 1.62 ± 0.05 meV) of the Gold nanoparticle-assembled mycelium film fit well with network assemblies of oligonucleotide-functionalized gold nanoparticles described previously (Park, et al., The Electrical properties of gold nanoparticle assemblies linked by DNA. Angew. Chem. Int. Ed. 39, 3845-3848 (2000)). Thus, micrometer-structures and materials constructed by the methods of the present invention may have magnetic or semiconductive qualities. Alternatively, the microorganisms may be embedded in a resin allowing study of the metal nanoparticle-hyphae assembly. These studies show that the cylindrical shape of the nanoparticle assembly remains with nanoparticles adsorbed on the cell wall surface of the hypha to form a ring structure. The diameter of the ring is dependent upon the different microorganism species employed. For example, the use of Aspergillus niger results in a ring structure having a diameter of approximately 5.5 μm whereas the use of Mucor hiemalis produces a diameter of about 15 μm and Streptomyces venezuelae a diameter of about 0.5 μm. In one embodiment of the present invention, the building block used in the production of this microorganism-templated macrostrucrure is a metal particle having surface modifications that are displayed on the surface of the biotemplate. For example, gold particles loaded with thiolated ssDNA can be used to form a macrostrucrure displaying a specific DNA sequence or a variety of multiple DNA sequences. On average, there are about 100 oligonucleotide strands conjugated with each 13-nm gold nanoparticle. The micrometer structures constructed from these building blocks gain the sequence-specific recognition property of the chosen DNA strand. For example, microorganisms can be loaded with 13 nm gold particles that have been functionalized with 5' thiolated oligonucleotide strands having the sequence: 5'- AlOAATATTGATAAGGAT-3' (SEQ ID NO: 1). At 45°C, the cottony mass of these DNA-loaded nanoparticles will not significantly absorb 30 nm gold nanoparticles modified with a non-complementary 5' thiolated oligonucleotide strand having the sequence: 5' -AIOTAACAATAATCCCTC- 3' (SEQ ID NO: 2). Under the same conditions however, the mass selectively absorbs 30 nm gold nanoparticles loaded with 5' thiolated oligonucleotide strands having the complementary sequence: 5'- A10ATCCTTATCAATATT-3' (SEQ ID NO: 3). Similarly, the method of the present invention can be used with gold nanoparticles having other surface modifications such as, BSBP (Bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt). Referring to Figures 3b and 3 c, the section images show that hybridizing 30 nm nanoparticles with DNA strands on the surface of hyphae loaded with 13 nm nanoparticles with the complementary DNA strand yields two layers of different sized nanoparticles (13 nm, 30 nm) arranged on the surface of the hyphae. As shown in the section images in Figure 4b and 4c, this results a double-layer nanoparticle ring with 13 nm particles inside and 30 nm particles outside.

The advantages of using various microorganisms (e. g. virus, bacteria, and fungi) as "bio-templates" for microscopic structure construction are the unique structural motifs that can be formed based on the biological properties of the individual species selected. Additionally, these microorganisms reproduce their structures relatively fast and organize their motifs, sometimes in a orderly fashion with a characteristic length scale.

This application claims priority under 35 USC § 119(e) to U.S. Provisional Application No. 60/403,991, filed August 16, 2002, which is incorporated herein in its entirety by this reference.

Claims

What is Claimed is:

I . A method of fabricating microstructures comprising: a. inoculating a growth media containing metal nanoparticles with a microorganism, b. maintaining the microorganism inoculated growth media under conditions which allow growth of the microorganism and accumulation of the metal nanoparticles by the microorganism; and, c. treating the microorganisms containing metal nanoparticles to form a microstructure of metal nanoparticles templated from the microorganism.

2. The method of Claim 1, wherein the metal is selected from the group consisting of gold and silver.

3. The method of Claim 1, wherein the metal nanoparticle has a diameter between about 5 inn and about 100 nm.

4. The method of Claim 1, wherein the metal nanoparticle is a gold nanoparticle having a diameter between about 10 nm and about 40 nm.

5. The method of Claim 1, wherein the metal nanoparticle is a gold nanoparticle having a diameter of about 13 nm.

6. The method of Claim 1, wherein the metal nanoparticle is a gold nanoparticle having a diameter of about 30 nm.

7. The method of Claim 1, wherein the microorganism is a rod or sphere- shaped microorganism.

8. The method of Claim 7, wherein the microorganism is fungal or bacterial.

9. The method of Claim 7, wherein the microorganism is selected from the group consisting of Penicilliun notatum, Rhizopus stolonifer, Mucor hiemalis, Streptomyces venezuelae and Aspergillus niger.

10. The method of Claim 1, wherein the growth media is 0.5XM9 minimal media.

II. The method of Claim 1, wherein the metal nanoparticles have a concentration of about 7 nM in the growth media.

12. The method of Claim 1, wherein the step of treating the microorganisms containing metal nanoparticles comprises air-drying the microorganisms.

13. The method of Claim 1, wherein the step of treating the microorganisms containing metal nanoparticles comprises contacting the microorganisms with acetone.

14. The method of Claim 1, wherein the step of treating the microorganisms containing metal nanoparticles comprises embedding the microorganisms in resin.

15. The method of Claim 1, wherein the metal nanoparticle has surface modifications.

16. The method of Claim 15, wherein the surface modifications are chosen from the group consisting of thiolated ohgonucleotides and Bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt.

17. The method of Claim 1, wherein the metal nanoparticle has been modified to contain thiolated first ohgonucleotides on a surface of the nanoparticle and the method comprises the additional step of hybridizing second ohgonucleotides having a complementary sequence to the sequence of the thiolated first ohgonucleotides to the thiolated first ohgonucleotides.

18. A rod or sphere shaped microorganism with a metal nanoparticle having a surface modification accumulated on a surface of the microorganism.

19. The rod or sphere shaped microorganism of Claim 18, wherein the surface modification is a thiolated oligonucleotide.

20. The rod or sphere shaped microorganism of Claim 18, wherein the surface modification is Bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt.

21. The rod or sphere shaped microorganism of Claim 18, wherein the microorganism is fungal or bacterial.

22. The rod or sphere shaped microorganism of Claim 18, wherein the microorganism is selected from the group consisting of Penicilliun notatum, Rhizopus stolonifer, Mucor hiemalis, Streptomyces venezuelae and Aspergillus niger.

23. The rod or sphere shaped microorganism of Claim 18, wherein the metal is selected from the group consisting of gold and silver.

24. The rod or sphere shaped microorganism of Claim 18, wherein the metal nanoparticle has a diameter between about 5 nm and about 100 nm.

25. The rod or sphere shaped microorganism of Claim 18, wherein the metal nanoparticle is a gold nanoparticle having a diameter between about 10 nm and about 40 nm.

26. The rod or sphere shaped microorganism of Claim 18, wherein the metal nanoparticle is a gold nanoparticle having a diameter of about 13 nm.

27. The rod or sphere shaped microorganism of Claim 18, wherein the metal nanoparticle is a gold nanoparticle having a diameter of about 30 nm.

28. The rod or sphere shaped microorganism of Claim 18, wherein the microorganisms have been air-dried.

29. The rod or sphere shaped microorganism of Claim 18, wherein the microorganisms have been dehydrated with acetone.

30. The rod or sphere shaped microorganism of Claim 18, wherein the microorganisms have been embedded in resin.