Rapid Gene Sensors from Carbon Nanotube-DNA Systems

Abstract

Methods, devices, and/or systems for providing carbon nanotube material that interacts with nucleotides to form CNT-nucleotide nanostructures wherein the CNT-nucleotide nanostructures form detectable network structures upon reactions with nucleic acids having targeted sequences.

Claims

1. A carbon nanotube probe comprising a functionalized carbon nanotube coupled to one or more nucleic acid probes that bind a target, wherein two or more carbon nanotube probes associate in the presence of the target forming a carbon nanotube network comprising a plurality of carbon nanotube probes and a plurality of targets.

2. The composition of claim 1, wherein the nucleic acid probe is non-covalently bound to the carbon nanotube.

3. The composition of claim 1, wherein the nucleic acid probe is covalently bound to the carbon nanotube.

4. The composition of claim 1, wherein the nucleic acid probe is DNA, RNA, or DNA and RNA.

5. The composition of claim 1, wherein the nucleic acid probe is at least 20 nucleotides.

6. The composition of claim 1, wherein the nucleic acid probe is at least 50 nucleotides.

7. The composition of claim 1, wherein the carbon nanotube is a single walled carbon nanotube.

8. The composition of claim 1, wherein the carbon nanotubes are 5 nm to 5 μm in length.

9. The composition of claim 1, wherein the carbon nanotubes have an outer diameter of 1 nm to 10 nm.

10. The composition of claim 1, wherein the carbon nanotube has a length to diameter ratio of 5 to 1,000,000.

11. The composition of claim 8, wherein the carbon nanotube is functionalized with at least one group defined as: ##STR00002##

12. A carbon nanotube network comprising a plurality of carbon nanotubes coupled to one or more nucleic acid probes and a plurality of targets, wherein association of the carbon nanotube probes induced by the target forms a carbon nanotube network.

13. The network of claim 12, wherein the target is a nucleic acid.

14. The network of claim 13, wherein the nucleic acid is a single stranded nucleic acid.

15. The network of claim 13, wherein the nucleic acid is DNA or RNA.

16. The network of claim 12, wherein the carbon nanotube network is a detectable carbon nanotube network.

17. The composition of claim 12, wherein the carbon nanotube network is a hydrogel or an aggregate.

18. The composition of claim 12, wherein the carbon nanotube network is detectable by bright field optical microscopy, resonance raman spectroscopy, differential pulse voltammetry, and/or dynamic light scattering.

19. A method for detecting a single-stranded or double-stranded nucleic acid having a target sequence comprising the steps of: (a) contacting a sample suspected of containing said nucleic acid having a target sequence with a carbon nanotube probe of claim 1, wherein the carbon nanotube probe is configured to form a network structure upon contact with a nucleic acid having a target sequence; (b) detecting the presence of the nucleic acid having a target sequence by detecting a carbon nanotube probe network structure that is formed in the presence of a target nucleic acid.

20. A process for making a carbon nanotube probe composition comprising: (a) functionalizing a carbon nanotube with a functional group that is capable of binding at least one nucleotide and/or nucleic acid; (b) contacting the functionalized carbon nanotube with at least one nucleotide and/or nucleic acid wherein the at least one nucleotide is configured to bind a nucleic acid having a target sequence; (c) sonicating the solution containing the functionalized carbon nanotube and at least one nucleotide and/or nucleic acid.

Description

DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0022] FIG. 1. Sequence-dependent aggregation (network structure) of single-wall carbon nanotubes-single stranded DNA (SWCNT-ssDNA) nanostructures.

[0023] FIG. 2(a) Chemical structure of functionalized SWCNTs 1-5. (b) Bright field optical microscopy of 1. (c) Resonance Raman of 1 reveals CNT-specific absorbance. (d) Dynamic light scattering of 1 in aqueous media indicate homogeneous dispersion in solution.

[0024] FIG. 3 Sequence-dependent aggregation of SWCNT(1).

[0025] FIG. 4 Real-time gene sensing technology of infectious diseases.

DESCRIPTION

[0026] Carbon nanotubes (CNTs) are allotropes of carbon with a cylindrical nanostructure. CNTs are members of the fullerene structural family. Their name is derived from their long, hollow structure with the walls formed by one-atom-thick sheets of carbon, called graphene. These sheets are rolled at specific and discrete (“chiral”) angles, and the combination of the rolling angle and radius decides the nanotube properties; for example, whether the individual nanotube shell is a metal or semiconductor. Nanotubes are categorized as single-walled nanotubes (SWNTs) and multi-walled nanotubes (MWNTs). Nanotubes have been constructed with length-to-diameter ratio of up to 132,000,000:1, significantly larger than for any other material. These cylindrical carbon molecules have unusual properties, which are valuable for nanotechnology, electronics, optics and other fields of materials science and technology.

[0027] Embodiments are directed to compositions, devices, and methods of using and making of CNT material that interacts with nucleotides or nucleic acids such as DNA to form CNT-nucleotide nanostructures that are capable of forming network structures, such as hydrogels or arrogates, upon binding or aggregation with nucleic acid(s) having a target sequence. In some instances, this technology works by means of designed nanostructures that polymerize or aggregate upon binding with a target nucleic acid. In some instances, the polymerization of the composition is sequence specific to the nucleic acid having a target sequence.

[0028] In some aspects, the network structure may be detected by direct or indirect visualization. In one aspect the network structure is detected by bright field optical microscopy, resonance raman, dynamic light scattering, and/or differential pulse voltammetry, etc. In a further aspect detection can be determined by a change in viscosity or gelling of a probe solution.

[0029] In some aspects, the CNT-nucleotide material forms a network structure when in contact with nM or lower concentrations of nucleic acids with target sequences. In one instance, the CNT-nucleotide may detect in real time the presence of nucleic acids with target sequences.

[0030] Compositions and methods described herein can be applied in many fields, including medical diagnostics and microbe detection. In some instances, compositions and methods described herein can be applied in real-time nucleic acids sensors and in nanofluidics based rapid diagnostic technologies.

[0031] FIG. 1 provides a non-limiting example of this approach. Single-wall carbon nanotubes (SWCNTs) and DNA materials can be used in real-time detection of specific nucleic acid sequences. In one instance, this new technology works by means of designed nanostructures that polymerize or aggregate upon reaction with targeted nucleic acids as shown in FIG. 1. Aggregation of the nanostructures induced by a nucleic acid having a specific sequence may then be detected by bright field optical microscopy, resonance raman, dynamic light scattering, and/or differential pulse voltammetry, etc.

[0032] Embodiments are directed to carbon nanotube probe compositions and related methods for detecting a variety of pathogens or potential pathogens (e.g., NIAID Category A, B, and C priority pathogens). In particular aspects of the invention the compositions and methods of the invention may be used to detect a biological weapon or opportunistic microbe.

[0033] There are numerous microbes that are considered pathogenic or potentially pathogenic under certain conditions (i.e., opportunistic pathogens/microbes). Bacterial microbes that can be detected using compositions and methods described herein include, but are not limited to various species of the Bacillus, Yersinia, Franscisella, Streptococcus, Staphylococcus, Pseudomonas, Mycobacterium, Burkholderia genus of bacteria. Particular species of bacteria that can be detected include, but is not limited to Bacillus anthracis, Yersinia pestis, Francisella tularensis, Streptococcus pnemoniae, Staphylococcus aureas, Pseudomonas aeruginosa, Burkholderia cepacia, Corynebacterium diphtherias, Clostridia spp, Shigella spp., and Mycobacterium avium.

[0034] There are numerous viruses and viral strains that can be detected using the compositions or methods described herein. Viruses can be placed in one of the seven following groups: Group I: double-stranded DNA viruses, Group II: single-stranded DNA viruses, Group III: double-stranded RNA viruses, Group IV: positive-sense single-stranded RNA viruses, Group V: negative-sense single-stranded RNA viruses, Group VI: reverse transcribing Diploid single-stranded RNA viruses, Group VII: reverse transcribing Circular double-stranded DNA viruses. Viruses include the family Adenoviridae, Arenaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae (Alphaherpesvirinae, Betaherpesvirinae, Gammaherpesvirinae), Nidovirales, Papillomaviridae, Paramyxoviridae (Paramyxovirinae, Pneumovirinae), Parvoviridae (Parvovirinae, Picornaviridae), Poxyiridae (Chordopoxyirinae), Reoviridae, Retroviridae (Orthoretrovirinae), and/or Togaviridae. These virus include, but are not limited to various strains of influenza, such as avian flu (e.g., H5N1). Particular virus from which a subject may be protected include, but is not limited to Cytomegalovirus, Respiratory syncytial virus and the like. Examples of pathogenic virus that can be detected include, but are not limited to Influenza A, H5N1, Marburg, Ebola, Dengue, Severe acute respiratory syndrome coronavirus, Yellow fever virus, Human respiratory syncytial virus, Vaccinia virus and the like.

[0035] There are numerous fungal species that are considered pathogenic or potentially pathogenic under certain conditions that can be detected using the compositions and methods described herein. Fungi include, but are not limited to Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Pneumocystis carinii, and Blastomyces dermatitidis.

[0036] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Preparation and Characterization of Functionalized CNTs

[0037] Functionalized CNTs—Several functionalized single-wall carbon nanotubes (SWCNTs) (1-5) have been prepared and characterized, FIG. 2(a), following established synthetic protocols (Mickelson et al., 1999; Bahr et al., 2001; Holzinger et al., 2001; Georgakilas et al., 2002). These functionalized SWCNTs (1-5) were shown to be capable of making stable suspensions in in aqueous media while increasing their affinity to bind DNA.

[0038] Characterization of CNTs—Optical microscopy, resonance raman, and dynamic light scattering were used to determine the stability in water and dispersion in water of the functionalized SWCNTs. It was determined that the functionalized SWCNTs form stable homogenous dispersions in water suitable for further reactions with DNA. FIG. 2(b)-FIG. 2(d) shows data results of SWCNT(1) characterization, which is representative of SWCNT's (2)-(5).

EXAMPLE 2

Preparation and Characterization of CNT-Nucleotides

[0039] CNT-nucleotides—Functionalized SWCNT(1) was reacted with single stranded DNA composed of C.sub.12A.sub.12, where C=cytosine and A=adenine, by sonication in the presence of the single-stranded DNA according to the methods described in Zheng et al., Nat. Mater. 2003 and Zheng et al., Science 2003. The product of the reaction was a SWCNT bound by the C.sub.12A.sub.12 nucleotide (1-DNA(C.sub.12A.sub.12)).

[0040] Characterization of CNT-nucleotides—The network structure of 1-DNA(C.sub.12A.sub.12) was determined by dynamic light scattering. 1-DNA(C.sub.12A.sub.12) forms stable bundles of discrete sizes similar to those illustrated in FIG. 1 (top, middle section).

EXAMPLE 3

Sensing Nucleic Acids Using CNT-Nucleotide

[0041] The network structure of a CNT-nucleotide was determined with and without the presence of a target nucleic acid. The network structure of the following solutions were determined by dynamic light scattering: 1-DNA(C.sub.12A.sub.12) solution of Example 2; 1-DNA(C.sub.12A.sub.12) exposed to a target single stranded DNA composed of T24 (DNA(T.sub.12)), where T=thymine; and 1-DNA(C.sub.12A.sub.12) exposed to a non-target control single strand DNA composed of A.sub.12 (DNA((A.sub.12)), where A=adenine. It was determined that aggregation is only triggered by the targeted DNA sequence (FIG. 3). It was also determined that aggregation occurred when the CNT-nucleotide was contacted with target nucleic acid at nM concentrations.

EXAMPLE 4

Sensing Nucleic Acids S Using SWCNT-ssDNA

[0042] SWCNT-nucleotide technology as described herein can be used for the real-time sensing of conserved single-stranded RNA from influenza virus, for example. The SWCNT-nucleotide technology can be directed to highly conserved regions of virus as the target sequence. An example of application of this technology is outlined in FIG. 4.

[0043] The technology described herein can be used as real-time gene sensors of nucleic acids such as those of infectious diseases. It is further contemplated that this technology can be used in the context of nanofluidics for rapid diagnosis in medical applications.

REFERENCES

[0044] Bahr et al., J. Am. Chem. Soc. 2001, 123, 6536. [0045] Ferguson et al., Nat. Biotechnol.,1996, 14, 1681. [0046] Georgakilas et al., Chem. Commun. 2002, 3050. [0047] Holzinger et al., Angew. Chem., Int. Ed. 2001, 40, 4002. [0048] Mickelson et al., J. Phys. Chem. B 1999, 103, 4318. [0049] Taylor and Schultz, Handbook of Chemical and Biological Sensors. Institute of Physics Publishing, Bristol, UK, 1996. [0050] Zhai et al., Biotechnol. Adv.,1997, 15, 43. [0051] Zheng et al., Nat. Mater. 2003, 2, 338. [0052] Zheng et al., Science 2003, 302, 1545.