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The protein–nanomaterial interface
The protein–nanomaterial interface Prashanth Asuri, Shyam Sundhar Bale, Sandeep S Karajanagi and Ravi S Kane Developments in the past few years have illustrated the potentially revolutionizing impact of nanomaterials, especially in biomedical imaging, drug delivery, biosensing and the design of functional nanocomposites. Methods to effectively interface proteins with nanomaterials for realizing these applications continue to evolve. Proteins are being used to control both the synthesis and assembly of nanomaterials. There has also been an increasing interest in understanding the influence of nanomaterials on the structure and function of proteins. Understanding and controlling the protein–nanomaterial interface will be crucial for designing functional protein– nanomaterial conjugates and assemblies. Addresses Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA Corresponding author: Kane, Ravi S ([email protected])
Current Opinion in Biotechnology 2006, 17:562–568 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 2nd October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.09.002
Introduction There has been considerable progress in the synthesis of nanomaterials with precise dimensions, geometries, and surface properties [1,2], and there is now an increasing interest in understanding and controlling the interactions of nanomaterials with biological molecules such as proteins [3,4,5,6]. Proteins have been used to functionalize nanomaterials and to influence their properties for applications ranging from sensing [7,8,9] and diagnostics [10,11] to delivery [12,13], and for the design of nanocomposites [14–17] (see also Update). Nanomaterial properties in turn have a strong influence on the structure and function of proteins, and there has been increasing emphasis on obtaining a fundamental understanding of these effects [4,5,6,18,19,20]. This review focuses on recent advances in understanding and applying protein–nanomaterial interactions, with particular emphasis on manuscripts published after 2003 (Figure 1). Current Opinion in Biotechnology 2006, 17:562–568
Protein structure and function on nanomaterials Although there have been numerous attempts to interface proteins with nanomaterials, many of these studies have focused on the modification and/or enhancement of nanomaterial properties to confer a specific biological function. It is, however, also important to understand how nanomaterial properties such as curvature and surface chemistry influence the structure and function of conjugated proteins [4,5,18,19,20,21]. Vertegel et al. [18] and Lundqvist et al. [19] studied proteins adsorbed onto silica nanoparticles of varying sizes and demonstrated that differences in nanoparticle size strongly influence the secondary structure and activity of adsorbed proteins. These studies indicated that smaller nanoparticles, perhaps owing to higher surface curvature, promoted the retention of native-like protein structure and function when compared with larger particles, at least for the proteins studied (i.e. lysozyme [18] and human carbonic anhydrase [19]). In a recent study, Roach et al. studied the effects of curvature on two structurally different proteins — bovine serum albumin and fibrinogen [4]. Although albumin retained more native-like structure on smaller particles, consistent with the previous work of Vertegel et al. [18] and Lundqvist et al. [19], fibrinogen was denatured to a greater extent on smaller particles; the influence of surface curvature on the structure of an adsorbed protein therefore seems to depend on the nature of the protein. A similar ‘protein-dependent’ behavior was reported by Karajanagi et al. [6] on single-walled carbon nanotubes (SWNTs). Spectroscopic measurements in conjunction with kinetic analysis revealed that soybean peroxidase (SBP) retained more of its native structure and activity when adsorbed onto SWNTs than chymotrypsin, which exhibited a nearly complete loss in activity and structure. The surface chemistry of a nanoparticle also influences the structure and function of adsorbed proteins. Roach et al. [4] reported a greater change in the secondary structure of both bovine serum albumin and fibrinogen on hydrophobic silica spheres than on hydrophilic ones. Moreover, Rotello and coworkers [5,22] demonstrated the ability to control protein structure and function by tailoring the surface chemistry of nanoparticles. By controlling the surface chemistry, they achieved three distinct levels of interaction of chymotrypsin with CdSe nanoparticles: no interaction (i.e. no binding to the nanoparticles); enzyme inhibition with denaturation; and enzyme inhibition with retention of structure [5] (see also Update). www.sciencedirect.com
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Figure 1
Understanding and controlling the protein–nanomaterial interface. Areas of interest include understanding how protein structure and function is affected by attachment to nanomaterials, using proteins to control the assembly of nanomaterials, and applications of protein–nanomaterial conjugates.
Finally, Asuri and colleagues [20,23] have uncovered a novel property of SWNTs — their ability to stabilize proteins under harsh conditions to a greater extent than conventional flat supports. For instance, the half-life of SBP adsorbed onto SWNTs at 95 8C was 90 min, 10fold greater than that of the native enzyme and 1.9 times that of SBP adsorbed on graphite flakes or other flat supports (Figure 2a). Moreover, the enhanced stabilization on SWNTs was not unique to SBP and was also seen for the unrelated protease, subtilisin Carlsberg. Experimental and theoretical analyses suggested that lateral interactions between adjacent adsorbed proteins contribute to protein deactivation in harsh environments and that these unfavorable interactions are suppressed on highly curved supports such as SWNTs relative to flat surfaces (Figure 2b) [20]. This work also suggests that enhancements in protein stability should not be unique to SWNTs, and could be obtained with other nanomaterials; enhanced protein stability on nanomaterials might therefore be a widely applicable phenomenon. However, application of this phenomenon requires that a protein retain significant activity during the initial adsorption step, a condition that is satisfied by several, but not all, proteins. www.sciencedirect.com
Figure 2
Protein behaviour on single-walled carbon nanotubes (SWNTs). (a) Time-dependent loss of activity of soybean peroxidase (SBP) adsorbed onto SWNTs (black circles), SBP adsorbed onto graphite flakes (grey squares), and native SBP (open circles) at 95 8C (data taken from [20]). (b) Schematic (drawn approximately to scale) depicting SBP molecules adsorbed onto a flat support (left) and on a nanoscale cylindrical support (right). The yellow line indicates the distance between adjacent proteins along the protein–substrate interface; for the same value of this average separation, curvature of the support might increase the average center-to-center distance and suppress unfavorable lateral interactions between adjacent proteins. (Part (a) was reproduced with permission from [20]. Copyright 2006 American Chemical Society.)
Collectively, these studies suggest that the structure, activity and stability of adsorbed proteins can be strongly influenced by both the surface chemistry of the nanomaterial and its curvature, but in a protein-dependent manner. Investigating the structure and function of proteins adsorbed onto different nanomaterials, as highlighted in this section, will be crucial for developing a better understanding of protein–material interactions at the nanoscale and for designing functional protein– nanomaterial conjugates.
Protein-mediated assembly of nanomaterials Although the protein- and peptide-mediated synthesis of nanomaterials is itself an exciting field of research [24–27], this topic is beyond the scope of this review. We focus here only on reports concerning the proteinmediated assembly of nanomaterials (Figure 3). For instance, biotin–streptavidin [16,28,29] and antigen– Current Opinion in Biotechnology 2006, 17:562–568
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Figure 3
Schematic representation of possible approaches for the protein-mediated assembly of nanoparticles, nanotubes and nanowires (Figure inspired by [28–32]). (a) One-dimensional assembly of nanoparticles, (b) two-dimensional or three-dimensional assembly of nanoparticles, and (c) end-to-end assembly of nanoparticles and nanotubes or nanowires. Red and yellow circles represent different nanoparticles and the grey cylinders represent nanotubes or nanowires. The black elements represent proteins or their ligands.
antibody [30,31] interactions have been used to direct the assembly of nanoparticles. In addition to spherical nanoparticles, anisotropic particles such as nanowires, nanotubes and M13 viruses have also been assembled using biomolecular recognition. For example, Searson and coworkers [32] directed the end-to-end assembly of Au/Pt/Au multisegment nanowires using biotin–avidin linkages. Similarly, Caswell et al. [33] reported that biotin-functionalized gold nanorods can be preferentially assembled in an end-to-end fashion using a streptavidin linker. Biotinstreptavidin interactions were also employed by Smorodin and colleagues [29] to assemble carbon nanotubes and gold nanoparticles. In a different approach, Belcher and coworkers [34] genetically modified M13 viruses to display a streptavidin-binding peptide and a hexahistidine peptide at opposite ends and demonstrated the assembly of the viruses into nanorings by adding a streptavidin-NiNTA linker. More recently, Yoo et al. [35] showed that the protein coat of the M13 virus could be tuned to manipulate the surface charge of the virus and hence its assembly on polyelectrolyte multilayers. Moreover, the monolayers of assembled viruses were used as scaffolds to nucleate, grow and align nanoparticles over multiple length scales. The loss or retention of the native structure of proteins upon their adsorption onto nanoparticles provides an additional variable for controlling nanoparticle assembly — interparticle spacing. Rotello and coworkers [36] used two different proteins to mediate the assembly of Current Opinion in Biotechnology 2006, 17:562–568
nanoparticles — chymotrypsin (which denatured upon binding) and cytochrome c (which retained its native structure) — and demonstrated that the nanoparticle spacing was controlled by protein conformation. Another exciting development in protein-mediated assembly involves the active and directed transport of materials by motor proteins such as kinesin. Researchers have demonstrated the assembly of CdSe quantum dots using microtubules and the transport of the resulting assemblies using kinesin [37]. Although the ability to transport cargoes using biomolecular shuttles has been reported earlier [38], the authors present a unique system wherein microtubules assemble nanoparticles into functional composites that can subsequently be actively transported to synthetic interfaces using kinesin. The bottom-up approach to designing functional nanocomposites involves the synthesis of nanomaterials of defined properties and their subsequent assembly into functional architectures. Proteins are particularly wellsuited for directing the assembly of nanomaterials into desired macroscopic structures.
Applications of protein–nanomaterial conjugates The unique properties of nanomaterials in conjunction with the biorecognition abilities of proteins offer particularly exciting opportunities in molecular imaging [10,11,39], therapy [40,41], and biomolecule delivery www.sciencedirect.com
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Figure 4
Schematic representation of silicon nanowire (SiNW)- and SWNT-based nanosensors. The binding of (a) a protein, (b) an antibody or (c) enzymatic activity, leads to modulation of nanomaterial properties (Figure inspired by [8,9,45,47,48]).
[12,13,42]. For instance, tunable fluorescence, high quantum yields, and high resistance to photo- and chemical degradation make quantum dots highly appealing for applications in cellular labeling, deep tissue imaging, and as efficient fluorescence resonance energy transfer donors — topics that have been reviewed previously [11]. Nanoshells are another class of novel nanomaterials that have shown promise in a variety of biomedical applications including whole-blood immunoassays, cancer therapy, and molecular imaging [10,40,43,44]. Hirsch et al. [44] demonstrated that nanoshells that strongly absorb light in the near infrared (NIR) can be used to selectively destroy solid tumors in vivo by delivering therapeutic dosages of heat via extracorporeally applied NIR radiation. Moreover, the nanoshells also provided enough scattering contrast to enable optical imaging. For instance, Drezek and coworkers [40] used immunotargeted nanoshells for the simultaneous imaging and destruction of breast carcinoma cells that overexpressed a clinically relevant cancer marker. Carbon nanotubes have also been employed for both biomolecule delivery and targeted therapy. Pantarotto et al. [12] demonstrated that carbon nanotubes functionalized with a peptide can translocate across the cell membrane of human and murine fibroblasts. Although the translocation mechanism of the carbon nanotubes was not clear, the authors suggested that the mechanism of nanotube uptake was not by endocytosis as the internalization was not affected by temperature or endocytosis inhibitors. Dai and coworkers [13] recently demonstrated that carbon nanotubes functionalized with proteins can enter nonadherent human cancer cells as well as adherent cell lines. The nanotubes could transport fluorescein-labeled streptavidin into cells, while the protein by itself did not enter cells. The authors reported that the uptake pathway of nanotubes into the cells was consistent with adsorption-mediated endocytosis. In conjunction with the aforementioned results obtained by Pantarotto www.sciencedirect.com
and colleagues, it is clear that further investigation is warranted to elucidate the mechanisms of cellular uptake of carbon nanotubes. Functionalized SWNTs can also be used to selectively target cancer cells. Irradiation of the nanotubes with NIR light after internalization into cells leads to the destruction of cancer cells [41]. Thus, the intrinsic optical and transport properties of nanotubes can lead to new opportunities in biomolecule delivery and cancer therapy. In addition to their applications in biomolecule delivery and targeted therapy, nanotube conjugates could also be used as vaccines. Pantarotto et al. [45] covalently attached an antigenic epitope from the foot-and-mouth disease virus to SWNTs; the resulting conjugates were immunogenic in vivo and could be used as vaccines. Another exciting class of applications utilizes the ability of proteins to modulate nanomaterial properties — a concept that is of particular use for designing biosensors (Figure 4) [9,46,47]. For example, Lieber and coworkers [48] demonstrated the use of biotinfunctionalized doped silica nanowires (SiNWs) to detect streptavidin at picomolar concentrations. The conductance of biotin-functionalized SiNWs increased rapidly upon exposure to streptavidin, consistent with the binding of a negatively charged species (streptavidin) to the boron-doped SiNW surface. More recently, Lieber and coworkers [49] demonstrated the label-free detection of small-molecule–protein interactions using SiNW sensors. Concentration-dependent binding of ATP and small-molecule inhibition of ATP binding to the tyrosine kinase Abl were characterized by monitoring the conductance of the SiNW devices. The SiNW-based label-free sensing strategy could be readily used for high-throughput screening and assay development. Carbon nanotubes have also been recently used for the detection of small molecules and proteins. Dai and coworkers [47] demonstrated the selective recognition of Current Opinion in Biotechnology 2006, 17:562–568
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monoclonal antibodies (mAbs) by a recombinant human antigen immobilized onto SWNTs. The binding of mAbs resulted in a decrease in the conductance of the SWNT devices, with a low detection limit (approximately nanomolar concentrations of mAbs). Interestingly, characterization of the nanotube devices revealed that the conductance change owing to protein adsorption originated primarily from the metal–nanotube contact and not from protein adsorption along the length of the nanotube [50]. This fundamental understanding will be important for optimizing the sensitivity and selectivity of nanotubebased biosensors. Finally, the enzymatic activity of adsorbed proteins might also enable the manipulation of nanomaterial properties. Strano and co-workers [46] have used SWNT–glucose-oxidase conjugates to develop glucose sensors. Glucose oxidase catalyzed the conversion of glucose to gluconolactone with hydrogen peroxide as the co-product; hydrogen peroxide reduced an electroactive moiety (ferricyanide) adsorbed on the SWNTs, thereby reversibly coupling the NIR fluorescence of SWNTs to the glucose concentration. The application of nanomaterial–bioconjugates as delivery vehicles, targeted therapeutic agents, and nanosensors represents only a small fraction of the exciting opportunities at the interface of nanotechnology and biotechnology. New applications continue to arise as the ability to manipulate these conjugates improves. For instance, Ipe et al. [51] recently demonstrated the design of quantum-dot–enzyme conjugates, wherein the superoxide and hydroxyl radicals generated by irradiating CdSe QDs at room temperature trigger enzyme activity. These results pave the way for designing novel nanosensors, photocatalysts for synthetic chemistry, and photosensitizers for intracellular reactions.
Conclusions The results reviewed here provide an excellent illustration of the symbiotic relationship that nanotechnology shares with biology. A better fundamental understanding of protein–nanomaterial interactions and the development of better methods to interface proteins with nanomaterials will undoubtedly have a major impact on biotechnology by providing new and improved sensing techniques, biocompatible, ‘smart’ and functional nanocomposites, highly effective drug delivery vehicles, and novel therapeutics.
Update In a recent study by Foo and colleagues [52], a biomimetic approach to synthesize silk-silica nanocomposites was developed using fusion proteins. In a further example of how the surface chemistry of nanoparticles can be used to control protein function, Bayraktar et al. [53] have designed functionalized gold nanoparticles that bind to cytochrome c or cytochrome c peroxidise and inhibit cytochrome c peroxidise activity. Current Opinion in Biotechnology 2006, 17:562–568
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Current Opinion in Biotechnology 2006, 17:562–568 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 2nd October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.09.002
Introduction There has been considerable progress in the synthesis of nanomaterials with precise dimensions, geometries, and surface properties [1,2], and there is now an increasing interest in understanding and controlling the interactions of nanomaterials with biological molecules such as proteins [3,4,5,6]. Proteins have been used to functionalize nanomaterials and to influence their properties for applications ranging from sensing [7,8,9] and diagnostics [10,11] to delivery [12,13], and for the design of nanocomposites [14–17] (see also Update). Nanomaterial properties in turn have a strong influence on the structure and function of proteins, and there has been increasing emphasis on obtaining a fundamental understanding of these effects [4,5,6,18,19,20]. This review focuses on recent advances in understanding and applying protein–nanomaterial interactions, with particular emphasis on manuscripts published after 2003 (Figure 1). Current Opinion in Biotechnology 2006, 17:562–568
Protein structure and function on nanomaterials Although there have been numerous attempts to interface proteins with nanomaterials, many of these studies have focused on the modification and/or enhancement of nanomaterial properties to confer a specific biological function. It is, however, also important to understand how nanomaterial properties such as curvature and surface chemistry influence the structure and function of conjugated proteins [4,5,18,19,20,21]. Vertegel et al. [18] and Lundqvist et al. [19] studied proteins adsorbed onto silica nanoparticles of varying sizes and demonstrated that differences in nanoparticle size strongly influence the secondary structure and activity of adsorbed proteins. These studies indicated that smaller nanoparticles, perhaps owing to higher surface curvature, promoted the retention of native-like protein structure and function when compared with larger particles, at least for the proteins studied (i.e. lysozyme [18] and human carbonic anhydrase [19]). In a recent study, Roach et al. studied the effects of curvature on two structurally different proteins — bovine serum albumin and fibrinogen [4]. Although albumin retained more native-like structure on smaller particles, consistent with the previous work of Vertegel et al. [18] and Lundqvist et al. [19], fibrinogen was denatured to a greater extent on smaller particles; the influence of surface curvature on the structure of an adsorbed protein therefore seems to depend on the nature of the protein. A similar ‘protein-dependent’ behavior was reported by Karajanagi et al. [6] on single-walled carbon nanotubes (SWNTs). Spectroscopic measurements in conjunction with kinetic analysis revealed that soybean peroxidase (SBP) retained more of its native structure and activity when adsorbed onto SWNTs than chymotrypsin, which exhibited a nearly complete loss in activity and structure. The surface chemistry of a nanoparticle also influences the structure and function of adsorbed proteins. Roach et al. [4] reported a greater change in the secondary structure of both bovine serum albumin and fibrinogen on hydrophobic silica spheres than on hydrophilic ones. Moreover, Rotello and coworkers [5,22] demonstrated the ability to control protein structure and function by tailoring the surface chemistry of nanoparticles. By controlling the surface chemistry, they achieved three distinct levels of interaction of chymotrypsin with CdSe nanoparticles: no interaction (i.e. no binding to the nanoparticles); enzyme inhibition with denaturation; and enzyme inhibition with retention of structure [5] (see also Update). www.sciencedirect.com
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Figure 1
Understanding and controlling the protein–nanomaterial interface. Areas of interest include understanding how protein structure and function is affected by attachment to nanomaterials, using proteins to control the assembly of nanomaterials, and applications of protein–nanomaterial conjugates.
Finally, Asuri and colleagues [20,23] have uncovered a novel property of SWNTs — their ability to stabilize proteins under harsh conditions to a greater extent than conventional flat supports. For instance, the half-life of SBP adsorbed onto SWNTs at 95 8C was 90 min, 10fold greater than that of the native enzyme and 1.9 times that of SBP adsorbed on graphite flakes or other flat supports (Figure 2a). Moreover, the enhanced stabilization on SWNTs was not unique to SBP and was also seen for the unrelated protease, subtilisin Carlsberg. Experimental and theoretical analyses suggested that lateral interactions between adjacent adsorbed proteins contribute to protein deactivation in harsh environments and that these unfavorable interactions are suppressed on highly curved supports such as SWNTs relative to flat surfaces (Figure 2b) [20]. This work also suggests that enhancements in protein stability should not be unique to SWNTs, and could be obtained with other nanomaterials; enhanced protein stability on nanomaterials might therefore be a widely applicable phenomenon. However, application of this phenomenon requires that a protein retain significant activity during the initial adsorption step, a condition that is satisfied by several, but not all, proteins. www.sciencedirect.com
Figure 2
Protein behaviour on single-walled carbon nanotubes (SWNTs). (a) Time-dependent loss of activity of soybean peroxidase (SBP) adsorbed onto SWNTs (black circles), SBP adsorbed onto graphite flakes (grey squares), and native SBP (open circles) at 95 8C (data taken from [20]). (b) Schematic (drawn approximately to scale) depicting SBP molecules adsorbed onto a flat support (left) and on a nanoscale cylindrical support (right). The yellow line indicates the distance between adjacent proteins along the protein–substrate interface; for the same value of this average separation, curvature of the support might increase the average center-to-center distance and suppress unfavorable lateral interactions between adjacent proteins. (Part (a) was reproduced with permission from [20]. Copyright 2006 American Chemical Society.)
Collectively, these studies suggest that the structure, activity and stability of adsorbed proteins can be strongly influenced by both the surface chemistry of the nanomaterial and its curvature, but in a protein-dependent manner. Investigating the structure and function of proteins adsorbed onto different nanomaterials, as highlighted in this section, will be crucial for developing a better understanding of protein–material interactions at the nanoscale and for designing functional protein– nanomaterial conjugates.
Protein-mediated assembly of nanomaterials Although the protein- and peptide-mediated synthesis of nanomaterials is itself an exciting field of research [24–27], this topic is beyond the scope of this review. We focus here only on reports concerning the proteinmediated assembly of nanomaterials (Figure 3). For instance, biotin–streptavidin [16,28,29] and antigen– Current Opinion in Biotechnology 2006, 17:562–568
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Figure 3
Schematic representation of possible approaches for the protein-mediated assembly of nanoparticles, nanotubes and nanowires (Figure inspired by [28–32]). (a) One-dimensional assembly of nanoparticles, (b) two-dimensional or three-dimensional assembly of nanoparticles, and (c) end-to-end assembly of nanoparticles and nanotubes or nanowires. Red and yellow circles represent different nanoparticles and the grey cylinders represent nanotubes or nanowires. The black elements represent proteins or their ligands.
antibody [30,31] interactions have been used to direct the assembly of nanoparticles. In addition to spherical nanoparticles, anisotropic particles such as nanowires, nanotubes and M13 viruses have also been assembled using biomolecular recognition. For example, Searson and coworkers [32] directed the end-to-end assembly of Au/Pt/Au multisegment nanowires using biotin–avidin linkages. Similarly, Caswell et al. [33] reported that biotin-functionalized gold nanorods can be preferentially assembled in an end-to-end fashion using a streptavidin linker. Biotinstreptavidin interactions were also employed by Smorodin and colleagues [29] to assemble carbon nanotubes and gold nanoparticles. In a different approach, Belcher and coworkers [34] genetically modified M13 viruses to display a streptavidin-binding peptide and a hexahistidine peptide at opposite ends and demonstrated the assembly of the viruses into nanorings by adding a streptavidin-NiNTA linker. More recently, Yoo et al. [35] showed that the protein coat of the M13 virus could be tuned to manipulate the surface charge of the virus and hence its assembly on polyelectrolyte multilayers. Moreover, the monolayers of assembled viruses were used as scaffolds to nucleate, grow and align nanoparticles over multiple length scales. The loss or retention of the native structure of proteins upon their adsorption onto nanoparticles provides an additional variable for controlling nanoparticle assembly — interparticle spacing. Rotello and coworkers [36] used two different proteins to mediate the assembly of Current Opinion in Biotechnology 2006, 17:562–568
nanoparticles — chymotrypsin (which denatured upon binding) and cytochrome c (which retained its native structure) — and demonstrated that the nanoparticle spacing was controlled by protein conformation. Another exciting development in protein-mediated assembly involves the active and directed transport of materials by motor proteins such as kinesin. Researchers have demonstrated the assembly of CdSe quantum dots using microtubules and the transport of the resulting assemblies using kinesin [37]. Although the ability to transport cargoes using biomolecular shuttles has been reported earlier [38], the authors present a unique system wherein microtubules assemble nanoparticles into functional composites that can subsequently be actively transported to synthetic interfaces using kinesin. The bottom-up approach to designing functional nanocomposites involves the synthesis of nanomaterials of defined properties and their subsequent assembly into functional architectures. Proteins are particularly wellsuited for directing the assembly of nanomaterials into desired macroscopic structures.
Applications of protein–nanomaterial conjugates The unique properties of nanomaterials in conjunction with the biorecognition abilities of proteins offer particularly exciting opportunities in molecular imaging [10,11,39], therapy [40,41], and biomolecule delivery www.sciencedirect.com
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Figure 4
Schematic representation of silicon nanowire (SiNW)- and SWNT-based nanosensors. The binding of (a) a protein, (b) an antibody or (c) enzymatic activity, leads to modulation of nanomaterial properties (Figure inspired by [8,9,45,47,48]).
[12,13,42]. For instance, tunable fluorescence, high quantum yields, and high resistance to photo- and chemical degradation make quantum dots highly appealing for applications in cellular labeling, deep tissue imaging, and as efficient fluorescence resonance energy transfer donors — topics that have been reviewed previously [11]. Nanoshells are another class of novel nanomaterials that have shown promise in a variety of biomedical applications including whole-blood immunoassays, cancer therapy, and molecular imaging [10,40,43,44]. Hirsch et al. [44] demonstrated that nanoshells that strongly absorb light in the near infrared (NIR) can be used to selectively destroy solid tumors in vivo by delivering therapeutic dosages of heat via extracorporeally applied NIR radiation. Moreover, the nanoshells also provided enough scattering contrast to enable optical imaging. For instance, Drezek and coworkers [40] used immunotargeted nanoshells for the simultaneous imaging and destruction of breast carcinoma cells that overexpressed a clinically relevant cancer marker. Carbon nanotubes have also been employed for both biomolecule delivery and targeted therapy. Pantarotto et al. [12] demonstrated that carbon nanotubes functionalized with a peptide can translocate across the cell membrane of human and murine fibroblasts. Although the translocation mechanism of the carbon nanotubes was not clear, the authors suggested that the mechanism of nanotube uptake was not by endocytosis as the internalization was not affected by temperature or endocytosis inhibitors. Dai and coworkers [13] recently demonstrated that carbon nanotubes functionalized with proteins can enter nonadherent human cancer cells as well as adherent cell lines. The nanotubes could transport fluorescein-labeled streptavidin into cells, while the protein by itself did not enter cells. The authors reported that the uptake pathway of nanotubes into the cells was consistent with adsorption-mediated endocytosis. In conjunction with the aforementioned results obtained by Pantarotto www.sciencedirect.com
and colleagues, it is clear that further investigation is warranted to elucidate the mechanisms of cellular uptake of carbon nanotubes. Functionalized SWNTs can also be used to selectively target cancer cells. Irradiation of the nanotubes with NIR light after internalization into cells leads to the destruction of cancer cells [41]. Thus, the intrinsic optical and transport properties of nanotubes can lead to new opportunities in biomolecule delivery and cancer therapy. In addition to their applications in biomolecule delivery and targeted therapy, nanotube conjugates could also be used as vaccines. Pantarotto et al. [45] covalently attached an antigenic epitope from the foot-and-mouth disease virus to SWNTs; the resulting conjugates were immunogenic in vivo and could be used as vaccines. Another exciting class of applications utilizes the ability of proteins to modulate nanomaterial properties — a concept that is of particular use for designing biosensors (Figure 4) [9,46,47]. For example, Lieber and coworkers [48] demonstrated the use of biotinfunctionalized doped silica nanowires (SiNWs) to detect streptavidin at picomolar concentrations. The conductance of biotin-functionalized SiNWs increased rapidly upon exposure to streptavidin, consistent with the binding of a negatively charged species (streptavidin) to the boron-doped SiNW surface. More recently, Lieber and coworkers [49] demonstrated the label-free detection of small-molecule–protein interactions using SiNW sensors. Concentration-dependent binding of ATP and small-molecule inhibition of ATP binding to the tyrosine kinase Abl were characterized by monitoring the conductance of the SiNW devices. The SiNW-based label-free sensing strategy could be readily used for high-throughput screening and assay development. Carbon nanotubes have also been recently used for the detection of small molecules and proteins. Dai and coworkers [47] demonstrated the selective recognition of Current Opinion in Biotechnology 2006, 17:562–568
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monoclonal antibodies (mAbs) by a recombinant human antigen immobilized onto SWNTs. The binding of mAbs resulted in a decrease in the conductance of the SWNT devices, with a low detection limit (approximately nanomolar concentrations of mAbs). Interestingly, characterization of the nanotube devices revealed that the conductance change owing to protein adsorption originated primarily from the metal–nanotube contact and not from protein adsorption along the length of the nanotube [50]. This fundamental understanding will be important for optimizing the sensitivity and selectivity of nanotubebased biosensors. Finally, the enzymatic activity of adsorbed proteins might also enable the manipulation of nanomaterial properties. Strano and co-workers [46] have used SWNT–glucose-oxidase conjugates to develop glucose sensors. Glucose oxidase catalyzed the conversion of glucose to gluconolactone with hydrogen peroxide as the co-product; hydrogen peroxide reduced an electroactive moiety (ferricyanide) adsorbed on the SWNTs, thereby reversibly coupling the NIR fluorescence of SWNTs to the glucose concentration. The application of nanomaterial–bioconjugates as delivery vehicles, targeted therapeutic agents, and nanosensors represents only a small fraction of the exciting opportunities at the interface of nanotechnology and biotechnology. New applications continue to arise as the ability to manipulate these conjugates improves. For instance, Ipe et al. [51] recently demonstrated the design of quantum-dot–enzyme conjugates, wherein the superoxide and hydroxyl radicals generated by irradiating CdSe QDs at room temperature trigger enzyme activity. These results pave the way for designing novel nanosensors, photocatalysts for synthetic chemistry, and photosensitizers for intracellular reactions.
Conclusions The results reviewed here provide an excellent illustration of the symbiotic relationship that nanotechnology shares with biology. A better fundamental understanding of protein–nanomaterial interactions and the development of better methods to interface proteins with nanomaterials will undoubtedly have a major impact on biotechnology by providing new and improved sensing techniques, biocompatible, ‘smart’ and functional nanocomposites, highly effective drug delivery vehicles, and novel therapeutics.
Update In a recent study by Foo and colleagues [52], a biomimetic approach to synthesize silk-silica nanocomposites was developed using fusion proteins. In a further example of how the surface chemistry of nanoparticles can be used to control protein function, Bayraktar et al. [53] have designed functionalized gold nanoparticles that bind to cytochrome c or cytochrome c peroxidise and inhibit cytochrome c peroxidise activity. Current Opinion in Biotechnology 2006, 17:562–568
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