Báo cáo khoa học: Nanoparticle–enzyme hybrid systems for nanobiotechnology

M I N I R E V I E W

Nanoparticle–enzyme 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

[or quantum-dot

Biomolecule–nanoparticle (NP) (QD)] hybrid systems combine the recognition and biocatalytic properties of biomolecules with the unique electronic, optical, and catalytic features of NPs and yield com- posite materials with new functionalities. The biomolecule–NP hybrid sys- tems 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 exem- plified by the development of amperometric glucose sensors by the electri- cal contacting of redox enzymes by means of AuNPs, and the design of an optical glucose sensor by the biocatalytic growth of AuNPs. The biocata- lytic growth of metallic NPs is used to fabricate Au and Ag nanowires on surfaces. The fluorescence 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

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.

Enzyme–metal NP hybrids for biosensing and for the generation of nanostructures

Biomolecules such as proteins, antibodies, antigens and DNA exhibit comparable dimensions to metallic or semiconductor nanoparticles (NPs). Thus, by integ- rating 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 recog- nition and catalytic properties of biomolecules might be envisaged. Indeed, substantial progress has been accomplished in recent years in the use of biomole- cule–NP hybrid systems as functional units for nano- biotechnology, and several detailed review articles have summarized the different nanobiomolecular constructs and their potential applications [1–3].

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

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Redox enzymes lack direct electrical contact with elec- trodes because of the insulation of their active sites by the protein shell ‘wiring’ of redox [4]. The electrical enzymes with electrodes is the basis for the development of amperometric biosensors or biofuel cells [5–8]. 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 This review addresses recent advances in the devel- opment of enzyme–NP conjugates and their specific

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A

B

C

Fig. 1. Electrical contacting of redox enzymes with an electrode by the reconsti- tution of apoproteins on cofactor-functional- ized AuNPs associated with the electrode. (A) Bioelectrocatalytic activation of GOx by the reconstituted apo-GOx on the FAD-func- tionalized AuNPs. (B) Electrocatalytic anodic currents generated by the reconstituted GOx-electrode in the presence of variable concentrations of glucose. (C) Bioelectrocat- alytic activation of GDH by the reconstitu- tion of apo-GDH on the PQQ-functionalized AuNPs associated with the electrode. (B is reprinted with permission from Y. Xiao et al. Science 299, 1877–1881. Copyright 2003 AAAS [9].)

by ‘wired’ electrically (GDH) was

to only develop amperometric

This paradigm is general and can be applied to other cofactor-dependent enzymes. For example, the pyrrolo- quinoline quinone (PQQ)-dependent glucose dehydro- genase 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 biosensing electrodes, but also to tailor voltammetric and optical biosensing surfaces. By constructing the GOx-reconsti- tuted 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 preven- ted the electron flow 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].

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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 flavin 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 reconstitu- ted on the FAD-functionalized particles. The alignment of GOx on the particles through the reconstitution process, and the shortening of the electron transfer dis- tances by the NPs, led to an enzyme–NP hybrid archi- tecture 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 gen- erated by the electrode, the electron transfer rate from the biocatalyst to the electrode was estimated to be ket ¼ 5000 s)1. This exchange rate is about sevenfold higher than the rate of electron transfer to the native acceptor of GOx (O2). The efficient 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 import- ant for the preparation of sensitive and selective ampero- metric glucose sensors, but it enables tailoring of effective anodes for biofuel cells. 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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the signal read-out

concentration of for optical 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 biocata- lyzed oxidation of tyrosine to l-DOPA, a product that – to AuNPs [17]. Similarly, alkaline reduced AuCl4 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, 1,4- dihydronicotinamide adenine dinucleotide (phosphate), – or Cu2+) and depos- reduced metal salts (e.g. AuCl4 ited the metals on AuNP seeds, which acted as cata- lysts. The resulting particles enabled the optical [19] or electrochemical [20] detection of the substrates specific for the enzymes.

electrical (conductivity) [13] or electrochemical [14] bio- sensors. Only recently, however, it was discovered that different enzymes catalyze the reduction of metal salts to metallic NPs, or that enzymes catalyze the depos- ition of metals on NP seeds. The biocatalytic forma- tion of metallic NPs, or the growth of metallic NPs, may have immediate nanobiotechnological applica- tions, as the plasmon absorbance of the NPs could probe enzyme activities and their substrates. For exam- ple, GOx oxidizes glucose to gluconic acid with the concomitant formation of H2O2. The latter product – and acts as a reducing agent which reduces AuCl4 deposits metal on the AuNP seeds, which act as cata- lysts for the metallization process [15] (Fig. 2A). As the concentration of H2O2 is controlled by the concen- tration of glucose, the extent of the enlargement of the particles is determined by the concentration of the sub- strate. Figure 2B shows the absorbance changes of AuNPs deposited on glass surfaces upon their enlarge- ment in the presence of different concentrations of glu- cose. The plasmon absorbance of the NPs increases as the concentration of glucose increases, providing an

A

[23,24].

B

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

the substrates specific for

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The synthesis of metallic nanowires is one of the challenging topics in nanobiotechnology. Biomolecules, specifically proteins, were used as templates for the ‘bottom-up’ chemical deposition of metallic nanowires [21]. For example, the polymerization of AuNP-func- tionalized actin units led to the formation of actin fila- ments which, upon chemical enlargement of the NPs, yielded continuous nanowires exhibiting metallic con- ductivity [22]. Similarly, metallic nanowires were syn- thesized in hollow amyloid templates 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 nano- circuitry and for the preparation of patterned nano- structures. Different biocatalyst–NP 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 synthe- sis 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 200–300 nm (Fig. 3B). Similarly, alkaline phosphatase modified with AuNPs acted as ‘biocatalytic ink’ for the depos- ition of silver nanowires upon hydrolyzing p-amino- phenol phosphate (2) (Fig. 3C). With this biocatalyst, continuous Ag nanowires with heights of 30–40 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’, con- the different sisting of enzymes, allows the stepwise, orthogonal formation of metal nanowires composed of different metals and

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Fig. 3. (A) Dip-pen nanolithographic pattern- ing of the GOx–AuNPs ‘biocatalytic ink’ on Si surfaces and the biocatalyzed enlarge- ment of the NPs to an Au nanowire. (B) AFM images of the enzyme nanopattern (top) and the resulting Au nanowire (bot- tom). (C) Biocatalytic enlargement of alkaline phosphatase (AlkPh), modified with AuNPs, with an Ag shell by the biocatalyzed hydroly- sis of (2). (D) AFM image of two orthogonal- ly 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 bio- catalytic growth of the nanowires exhibits a self-inhibi- tion 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.

functionalized semiconductor QDs have been used as fluorescence labels for biorecognition events [27,28]. However, the use of semiconductor QDs to follow bio- catalytic reactions requires the application of photo- physical mechanisms, such as fluorescence 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 telo- merase-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 use of coupled enzyme–NP reactions for the patterning of nanostructures on surfaces was also dem- onstrated [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 carb- oxylic acid units, and the biocatalyzed hydroxylation of the tyramine units to the respective catechol deriv- ative encoded the ligand structure for the self-assembly of boronate-functionalized AuNPs or magnetic NPs on the encoded patterns through the formation of catechol–Fe2+ ⁄ 3+ complexes catechol–boronate or between the surface and the particles (Fig. 4).

Biomolecule–semiconductor NPs for biosensing

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Semiconductor NPs (or quantum dots, QDs) reveal unique size-controlled optical properties [26]. Indeed, The association properties of the maltose-binding protein and the development of a competitive maltose biosensor were studied by the application of a CdSe QD–maltose-binding protein hybrid [32] (Fig. 5A). A b-cyclodextrin–QSY-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. the hydrolytic functions of a series of Similarly,

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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 acid- functionalized AuNPs or Fe3+ ions, associ- ated with the magnetic NPs, act as linkers to the generated catechol ligands.

specific sequences

for cleavage by thrombin was linked to the QDs. Oxi- dation of the tyrosine residue by tyrosinase generated the o-quinone derivative of l-DOPA, which quenched the luminescence of the QD. The subsequent throm- bin-stimulated cleavage of the peptide and removal of the quinone quencher units regenerated the fluores- cence properties of the QDs. Photoexcitation of

proteolytic enzymes were followed by the application of QD reporter units and the FRET process as a read- out mechanism [33,34]. CdSe QDs were modified with for different proteases, peptide where quencher units were tethered to the peptide ter- mini. Within this assembly the fluorescence 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 fluorescence 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 fluorescence of the QD, hydrolytic scission of the dye and its corresponding removal restored the fluores- cence.

two enzymes,

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In a related study [35], the biocatalytic functions tyrosinase and thrombin, were of probed by CdSe ⁄ ZnS QDs. A tyrosine-methyl ester- terminated peptide that included the specific sequence semiconductor NPs not only the photogenerated yields luminescence probes, but electron–hole pair may also stimulate the generation of photocurrents. Generation of photocurrents by bio- molecule–NP conjugates has been demonstrated in sev- eral systems that included semiconductor NP–DNA conjugates [36] or semiconductor–NP–enzyme hybrid [37,38]. Cytochrome c-mediated biocatalytic systems 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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{Fluorescence graph in (A)

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 fluorescence 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 protease- mediated hydrolysis of the rhodamine Red-X-functionalized peptide (3). The decrease in the fluorescence of the dye and the corres- ponding increase in the fluorescence of the QDs upon interaction with different concentrations of collagenase are depicted on the right. reprinted by permission from Macmillan Publishers Ltd: Nat Mater 2, 630–638, 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 acti- –) by vation of the cytochrome c-mediated reduction of nitrate (NO3 nitrate reductase (NR). (C) The photocurrents generated by the bio- catalytic 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.)

that electrically

(Fig. 6A). Photoexcitation of

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analogy, the use of cytochrome c in its oxidized form – to enabled the bioelectrocatalytic reduction of NO3 – by nitrate reductase (NR), while generating a NO2 cathodic photocurrent (Fig. 6B). The transfer of con- duction-band electrons to the oxidized, heme-contain- ing cofactor generated reduced cytochrome c, and the transfer of electrons from the electrode to the valence- band of the NPs restored the ground-state of the NPs. The cytochrome c-mediated biocatalyzed reduction of – to nitrite then enabled the formation of the NO3 cathodic photocurrent. The photocurrents generated by the biocatalytic cascades at various concentrations through a dithiol linker, and thiopyridine units, acting as promoter units 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 activa- tion of the oxidation of lactate by lactate dehydroge- nase (LDH) proceeds while generating an anodic photocurrent the NPs resulted in the injection of the conduction-band elec- trons into the electrode and the concomitant oxidation the reduced cytochrome c by the valence-band of holes. The resulting oxidized cytochrome c then medi- ated the LDH-biocatalyzed oxidation of lactate. In

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of the different substrates are depicted in Fig. 6C. These results show that the photoelectrochemical func- tions of semiconductor NPs could be used to develop sensors for biocatalytic transformations.

In a different study [37], CdS was modified with ace- tylcholinesterase, 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 photocur- rent was controlled by the concentration of the sub- strate, 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 NP–enzyme hybrids has involved their use as active templates for the ‘bottom- up’ synthesis of nanowires and nanopatterns. At pre- sent, 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 NP–enzyme functional hybrids for the biocatalytic synthesis of devices seems feasible. The use of NP–biomolecule hybrid systems, specifically NP–enzyme assemblies, is in the early phases of development. The results already obtained promise exciting future developments in this area of nanobiotechnology.

Conclusions and perspectives

Acknowledgements

Our research on NP–enzyme hybrid systems is suppor- ted by the Israeli–German Program (DIP) and by the Ministry of Science, Israel.

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