MINIREVIEW

Nanoparticleenzyme hybrid systems for nanobiotechnology

Itamar Willner, Bernhard Basnar and Bilha Willner
Institute of Chemistry, The Hebrew University of Jerusalem, Israel

Keywords enzymes; nanoparticles; nanowires; quantum dots; semiconductors Correspondence I. Willner, Institute of Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Fax: +972 2 6527715 Tel: +972 2 6585272 E-mail: willnea@vms.huji.ac.il (Received 5 October 2006, accepted 20 November 2006) doi:10.1111/j.1742-4658.2006.05602.x

Biomoleculenanoparticle (NP) [or quantum-dot (QD)] hybrid systems combine the recognition and biocatalytic properties of biomolecules with the unique electronic, optical, and catalytic features of NPs and yield composite materials with new functionalities. The biomoleculeNP hybrid systems allow the development of new biosensors, the synthesis of metallic nanowires, and the fabrication of nanostructured patterns of metallic or magnetic NPs on surfaces. These advances in nanobiotechnology are exemplied by the development of amperometric glucose sensors by the electrical contacting of redox enzymes by means of AuNPs, and the design of an optical glucose sensor by the biocatalytic growth of AuNPs. The biocatalytic growth of metallic NPs is used to fabricate Au and Ag nanowires on surfaces. The uorescence properties of semiconductor QDs are used to develop competitive maltose biosensors and to probe the biocatalytic functions of proteases. Similarly, semiconductor NPs, associated with electrodes, are used to photoactivate bioelectrocatalytic cascades while generating photocurrents.

Introduction
Biomolecules such as proteins, antibodies, antigens and DNA exhibit comparable dimensions to metallic or semiconductor nanoparticles (NPs). Thus, by integrating biomolecules and NPs into hybrid conjugates, new functional chemical entities that combine the unique electronic, optical, and catalytic properties of metallic or semiconductor NPs with the unique recognition and catalytic properties of biomolecules might be envisaged. Indeed, substantial progress has been accomplished in recent years in the use of biomoleculeNP hybrid systems as functional units for nanobiotechnology, and several detailed review articles have summarized the different nanobiomolecular constructs and their potential applications [13]. This review addresses recent advances in the development of enzymeNP conjugates and their specic

applications for sensing and nanocircuitry design. Its aim is to introduce some facets of nanobiotechnology and, together with the other articles in this mini-review series, to highlight the broadness and perspectives of the topic.

Enzymemetal NP hybrids for biosensing and for the generation of nanostructures

Redox enzymes lack direct electrical contact with electrodes because of the insulation of their active sites by the protein shell [4]. The electrical wiring of redox enzymes with electrodes is the basis for the development of amperometric biosensors or biofuel cells [58]. When it was realized that the spatial separation between the active site and the electrode is due to the insulating protein shell, gold (Au) NPs (1.4 nm) were used as

Abbreviations AFM, atomic force microscope; FRET, uorescence resonance energy transfer; GDH, glucose dehydrogenase; GOx, glucose oxidase; LDH, lactate dehydrogenase; NP, nanoparticle; PQQ, pyrroloquinoline quinone; QD, quantum dot.

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Fig. 1. Electrical contacting of redox enzymes with an electrode by the reconstitution of apoproteins on cofactor-functionalized AuNPs associated with the electrode. (A) Bioelectrocatalytic activation of GOx by the reconstituted apo-GOx on the FAD-functionalized AuNPs. (B) Electrocatalytic anodic currents generated by the reconstituted GOx-electrode in the presence of variable concentrations of glucose. (C) Bioelectrocatalytic activation of GDH by the reconstitution of apo-GDH on the PQQ-functionalized AuNPs associated with the electrode. (B is reprinted with permission from Y. Xiao et al. Science 299, 18771881. Copyright 2003 AAAS [9].)

nanoelectrodes to shorten electron transfer distances and mediate charge transport [9] (Fig. 1A). The AuNP was linked to the Au electrode by a dithiol bridge, and N-aminoethyl avin adenine dinucleotide, amino-FAD (1), was linked to the particles. The FAD cofactor units were extracted from active glucose oxidase (GOx) to yield the apoprotein, and apo-GOx was then reconstituted on the FAD-functionalized particles. The alignment of GOx on the particles through the reconstitution process, and the shortening of the electron transfer distances by the NPs, led to an enzymeNP hybrid architecture that revealed electrical contacting with the electrode. This enabled the bioelectrocatalytic oxidation of glucose (Fig. 1B). With the knowledge of the surface coverage of the enzyme and the saturation current generated by the electrode, the electron transfer rate from the biocatalyst to the electrode was estimated to be ket 5000 s)1. This exchange rate is about sevenfold higher than the rate of electron transfer to the native acceptor of GOx (O2). The efcient electron transport originates from a single NP implanted into the protein structure. This method for the effective electrical contacting of GOx with the electrode is not only important for the preparation of sensitive and selective amperometric glucose sensors, but it enables tailoring of effective anodes for biofuel cells.

This paradigm is general and can be applied to other cofactor-dependent enzymes. For example, the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) was electrically wired by the reconstitution of apo-GDH on PQQ-functionalized AuNPs [10] (Fig. 1C). The charge transport from the redox center to the AuNP acting as a relay was used not only to develop amperometric biosensing electrodes, but also to tailor voltammetric and optical biosensing surfaces. By constructing the GOx-reconstituted AuNP nanostructure on an Au electrode by long-chain alkane dithiol bridging units, the AuNPs were charged by the bioelectrocatalytic process, yet the dithiol bridges acted as a tunneling barrier that prevented the electron ow to the electrode. The charging of the particles was followed by the voltage generated on the electrode, or by the surface plasmon resonance shifts of the surface resulting from the charging of the particles [11]. The biocatalytic growth of metallic NPs represents a further interesting direction in nanobiotechnology [12]. The catalytic deposition of metals on NP seeds is a common practice in microelectronics, known as the electroless deposition process of metals. The catalytic enlargement of metallic NPs by chemical means also found different applications in the development of
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electrical (conductivity) [13] or electrochemical [14] biosensors. Only recently, however, it was discovered that different enzymes catalyze the reduction of metal salts to metallic NPs, or that enzymes catalyze the deposition of metals on NP seeds. The biocatalytic formation of metallic NPs, or the growth of metallic NPs, may have immediate nanobiotechnological applications, as the plasmon absorbance of the NPs could probe enzyme activities and their substrates. For example, GOx oxidizes glucose to gluconic acid with the concomitant formation of H2O2. The latter product acts as a reducing agent which reduces AuCl4 and deposits metal on the AuNP seeds, which act as catalysts for the metallization process [15] (Fig. 2A). As the concentration of H2O2 is controlled by the concentration of glucose, the extent of the enlargement of the particles is determined by the concentration of the substrate. Figure 2B shows the absorbance changes of AuNPs deposited on glass surfaces upon their enlargement in the presence of different concentrations of glucose. The plasmon absorbance of the NPs increases as the concentration of glucose increases, providing an
A

Fig. 2. (A) Biocatalytic enlargement of AuNPs by the GOx-mediated oxidation of glucose, and the catalytic reduction of AuCl4 by H2O2. (B) Absorbance spectra of the enlarged AuNPs synthesized by the GOx-mediated reaction in the presence of various concentrations of glucose for a xed time interval of 10 min. (B was adapted with permission from [15]. Copyright 2005 American Chemical Society.)

optical read-out signal for the concentration of glucose. The growth of metallic NPs and the optical monitoring of the biocatalytic transformations was extended to other enzymes. Tyrosinase, a melanoma cancer cell biomarker [16], was assayed by the biocatalyzed oxidation of tyrosine to l-DOPA, a product that reduced AuCl4 to AuNPs [17]. Similarly, alkaline phosphatase hydrolyzed p-aminophenol phosphate to p-aminophenol, which reduced Ag+ to a silver shell on AuNPs [18]. Also, NAD(P)+-dependent enzymes, such as alcohol dehydrogenase, were used as biocatalysts for the growth of AuNPs. The reduced cofactor, 1,4dihydronicotinamide adenine dinucleotide (phosphate), reduced metal salts (e.g. AuCl4 or Cu2+) and deposited the metals on AuNP seeds, which acted as catalysts. The resulting particles enabled the optical [19] or electrochemical [20] detection of the substrates specic for the enzymes. The synthesis of metallic nanowires is one of the challenging topics in nanobiotechnology. Biomolecules, specically proteins, were used as templates for the bottom-up chemical deposition of metallic nanowires [21]. For example, the polymerization of AuNP-functionalized actin units led to the formation of actin laments which, upon chemical enlargement of the NPs, yielded continuous nanowires exhibiting metallic conductivity [22]. Similarly, metallic nanowires were synthesized in hollow amyloid templates [23,24]. In contrast with the use of proteins as passive templates for the growth of nanowires, one can use enzymes and NPs as active hybrid systems for the synthesis of nanocircuitry and for the preparation of patterned nanostructures. Different biocatalystNP conjugates were used as active templates for the biocatalytic synthesis of metallic nanowires [18]. GOx functionalized with AuNPs acted as biocatalytic ink for the active synthesis of Au nanowires (Fig. 3A). Its patterning on a Si surface by dip-pen nanolithography, followed by the glucose-mediated generation of H2O2 and the catalytic enlargement of the NPs, led to the formation of Au nanowires with heights in the range 200300 nm (Fig. 3B). Similarly, alkaline phosphatase modied with AuNPs acted as biocatalytic ink for the deposition of silver nanowires upon hydrolyzing p-aminophenol phosphate (2) (Fig. 3C). With this biocatalyst, continuous Ag nanowires with heights of 3040 nm were prepared. The active biocatalytic growth of the nanowires has several important advantages for the future manufacture of nanocircuits. The synthesis of metallic nanowires by the developing solution, consisting of the substrates specic for the different enzymes, allows the stepwise, orthogonal formation of metal nanowires composed of different metals and

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Fig. 3. (A) Dip-pen nanolithographic patterning of the GOxAuNPs biocatalytic ink on Si surfaces and the biocatalyzed enlargement of the NPs to an Au nanowire. (B) AFM images of the enzyme nanopattern (top) and the resulting Au nanowire (bottom). (C) Biocatalytic enlargement of alkaline phosphatase (AlkPh), modied with AuNPs, with an Ag shell by the biocatalyzed hydrolysis of (2). (D) AFM image of two orthogonally synthesized nanowires consisting of the GOx-generated Au nanowire and of the AlkPh-synthesized Ag nanowire. (Figure reprinted with permission from [18]. Copyright 2006 Wiley-VCH.)

controlled dimensions (Fig. 3D). Furthermore, the biocatalytic growth of the nanowires exhibits a self-inhibition mechanism, and upon coating of the protein by the metal no further enlargement occurs. This allows the dimensions of the nanowires to be controlled by the size of the biocatalytic templates. The use of coupled enzymeNP reactions for the patterning of nanostructures on surfaces was also demonstrated [25]. A molecular nanopattern was generated on a long-chain alkylsiloxane monolayer associated with the surface by the electrochemical oxidation of the methyl head groups to carboxylic acid residues, using a conductive atomic force microscope (AFM) tip. Tyramine was then covalently linked to the carboxylic acid units, and the biocatalyzed hydroxylation of the tyramine units to the respective catechol derivative encoded the ligand structure for the self-assembly of boronate-functionalized AuNPs or magnetic NPs on the encoded patterns through the formation of catecholboronate or catecholFe2+ 3+ complexes between the surface and the particles (Fig. 4). Biomoleculesemiconductor NPs for biosensing Semiconductor NPs (or quantum dots, QDs) reveal unique size-controlled optical properties [26]. Indeed,

functionalized semiconductor QDs have been used as uorescence labels for biorecognition events [27,28]. However, the use of semiconductor QDs to follow biocatalytic reactions requires the application of photophysical mechanisms, such as uorescence resonance energy transfer (FRET), that enable the dynamics of the enzymatic reactions to be followed [29]. Several reports have addressed the use of CdSe QDs to follow the biocatalyzed replication of DNA [30], the telomerase-induced telomerization of nucleic acid [30], and the scission of duplex DNA by DNase [31] using FRET reactions. Similarly, semiconductor QDs were integrated with proteins, and the hybrid systems enabled the real-time analysis of the binding properties or the catalytic functions of the proteins. The association properties of the maltose-binding protein and the development of a competitive maltose biosensor were studied by the application of a CdSe QDmaltose-binding protein hybrid [32] (Fig. 5A). A b-cyclodextrinQSY-9 dye conjugate resulted in the quenching of the luminescence of the QDs by the dye units. Addition of maltose displaced the quencher units, and this regenerated the luminescence function of the QDs. This method enabled the development of a competitive QD-based sensor for maltose in solution. Similarly, the hydrolytic functions of a series of
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Fig. 4. Biocatalytic activation of encoded, functional ligands on surfaces for the addressable deposition of nanostructures consisting of AuNPs or magnetic NPs. Tyrosinase is used to activate the hydroxyphenyl structure while boronic acidfunctionalized AuNPs or Fe3+ ions, associated with the magnetic NPs, act as linkers to the generated catechol ligands.

proteolytic enzymes were followed by the application of QD reporter units and the FRET process as a readout mechanism [33,34]. CdSe QDs were modied with peptide sequences specic for different proteases, where quencher units were tethered to the peptide termini. Within this assembly the uorescence of the QDs was quenched (Fig. 5B). The hydrolytic cleavage of the peptide resulted in the removal of the quencher units, and this restored the uorescence generated by the QDs. For example, collagenase was used to cleave the rhodamine Red-X dye-labeled peptide (3) linked to CdSe ZnS QDs. While the tethered dye quenched the uorescence of the QD, hydrolytic scission of the dye and its corresponding removal restored the uorescence. In a related study [35], the biocatalytic functions of two enzymes, tyrosinase and thrombin, were probed by CdSe ZnS QDs. A tyrosine-methyl esterterminated peptide that included the specic sequence
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for cleavage by thrombin was linked to the QDs. Oxidation of the tyrosine residue by tyrosinase generated the o-quinone derivative of l-DOPA, which quenched the luminescence of the QD. The subsequent thrombin-stimulated cleavage of the peptide and removal of the quinone quencher units regenerated the uorescence properties of the QDs. Photoexcitation of semiconductor NPs not only yields luminescence probes, but the photogenerated electronhole pair may also stimulate the generation of photocurrents. Generation of photocurrents by biomoleculeNP conjugates has been demonstrated in several systems that included semiconductor NPDNA conjugates [36] or semiconductorNPenzyme hybrid systems [37,38]. Cytochrome c-mediated biocatalytic processes were coupled to CdS NPs, and the direction of the resulting photocurrent could be controlled by the oxidation state of the cytochrome c mediator [38]. The CdS NPs were immobilized on an Au electrode

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Fig. 5. Application of semiconductor QDs for optical biosensing. (A) Application of CdSe QDs for the competitive assay of maltose using the maltose-binding protein as sensing material and QSY-9-CD as FRET quencher. The increase in the uorescence of the QDs upon analyzing increasing amounts of maltose is depicted on the right. (B) Application of CdSe ZnS QDs for the optical analysis of proteasemediated hydrolysis of the rhodamine Red-X-functionalized peptide (3). The decrease in the uorescence of the dye and the corresponding increase in the uorescence of the QDs upon interaction with different concentrations of collagenase are depicted on the right. {Fluorescence graph in (A) reprinted by permission from Macmillan Publishers Ltd: Nat Mater 2, 630638, copyright 2003 [32]. Fluorescence graph in (B) adapted with permission from [34]. Copyright 2006 American Chemical Society.}

Fig. 6. Generation of photocurrents by the photochemically induced activation of enzyme cascades by CdS NPs. (A) The photochemical activation of the cytochrome c-mediated oxidation of lactate in the presence of LDH (i.e. cytochrome b2). (B) The photochemical activation of the cytochrome c-mediated reduction of nitrate (NO3) by nitrate reductase (NR). (C) The photocurrents generated by the biocatalytic cascades in the presence of various concentrations of the substrates (lactate nitrate). (C is taken from [38]; reproduced by permission of the Royal Society of Chemistry.)

through a dithiol linker, and thiopyridine units, acting as promoter units that electrically communicate between the cytochrome c and the NPs, were linked to the semiconductor NPs (Fig. 6). In the presence of reduced cytochrome c, the photoelectrocatalytic activation of the oxidation of lactate by lactate dehydrogenase (LDH) proceeds while generating an anodic photocurrent (Fig. 6A). Photoexcitation of the NPs resulted in the injection of the conduction-band electrons into the electrode and the concomitant oxidation of the reduced cytochrome c by the valence-band holes. The resulting oxidized cytochrome c then mediated the LDH-biocatalyzed oxidation of lactate. In

analogy, the use of cytochrome c in its oxidized form enabled the bioelectrocatalytic reduction of NO3 to NO2 by nitrate reductase (NR), while generating a cathodic photocurrent (Fig. 6B). The transfer of conduction-band electrons to the oxidized, heme-containing cofactor generated reduced cytochrome c, and the transfer of electrons from the electrode to the valenceband of the NPs restored the ground-state of the NPs. The cytochrome c-mediated biocatalyzed reduction of NO3 to nitrite then enabled the formation of the cathodic photocurrent. The photocurrents generated by the biocatalytic cascades at various concentrations
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of the different substrates are depicted in Fig. 6C. These results show that the photoelectrochemical functions of semiconductor NPs could be used to develop sensors for biocatalytic transformations. In a different study [37], CdS was modied with acetylcholinesterase, and the biocatalyzed hydrolysis of thioacetylcholine generated thiocholine, which acted as electron donor for the photogenerated holes in the valence-band of the CdS NPs. The resulting photocurrent was controlled by the concentration of the substrate, and was depleted in the presence of inhibitors of acetylcholinesterase. This system has been suggested as a potential sensor for chemical warfare agents that act as inhibitors of acetylcholinesterase.

A further application of NPenzyme hybrids has involved their use as active templates for the bottomup synthesis of nanowires and nanopatterns. At present, the biocatalytic synthesis of nanowires is limited to the growth of metallic nanowires. One can envisage, however, the use of biocatalytic templates to synthesize polymer or semiconductor nanowires. Once these developments have materialized, the use of NPenzyme hybrids for the biocatalytic synthesis of functional devices seems feasible. The use of NPbiomolecule hybrid systems, specically NPenzyme assemblies, is in the early phases of development. The results already obtained promise exciting future developments in this area of nanobiotechnology.

Conclusions and perspectives

The unique chemical and physical properties of metallic or semiconductor NPs, together with the different nanotools to manipulate or pattern nanostructures on surfaces, add new dimensions to analytical science, lithographic nanostructuring, and the engineering of nanodevices. Not surprisingly, the use of biomoleculeNP hybrid systems has recently attracted immense scientic efforts, and ingenious electronic and optical biosensors have been assembled, new paradigms for the bottom-up construction of nanowires developed, and highly imaginative nanodevices fabricated. This article has addressed the specic use of enzymeNP hybrid systems in nanobiotechnology. The colorimetric assay of the activities of enzymes by the formation of metallic NPs suggests that new biosensor chips and supported biocatalytic optical sensors could be developed in the future. Furthermore, the size-controlled emission properties of semiconductor QDs and their use to probe enzyme functions suggest that, by the application of mixtures of QDs functionalized with different substrates, the multiplexed parallel analysis of the activities of different enzymes may be accomplished. The fact that NPs of enhanced structural and engineered complexity, which exhibit unique optical and electronic features, are continuously being synthesized and developed suggests that their coupling to biomolecules, specically enzymes, will lead to new sensor systems. For example, the biocatalytic growth of AuNPs in the shape of tripods or tetrapods by NAD+-dependent enzymes has enabled the optical detection of enzyme activities through longitudinal plasmon absorbance of the nanostructures [39]. Similarly, the coupling of graded arrays of QDs, which reveal intrastructure energytransfer cascades to enzymes, is expected to yield new functional sensors.
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Acknowledgements
Our research on NPenzyme hybrid systems is supported by the IsraeliGerman Program (DIP) and by the Ministry of Science, Israel.

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