Nanoparticle–enzyme hybrid systems

7/28/2019 Nanoparticleenzyme hybrid systems

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M I N I R E V I E W

Nanoparticleenzyme hybrid systems for

nanobiotechnology

Itamar Willner, Bernhard Basnar and Bilha Willner

Institute of Chemistry, The Hebrew University of Jerusalem, Israel

Introduction

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-

culeNP hybrid systems as functional units for nano-biotechnology, and several detailed review articles have

summarized the different nanobiomolecular constructs

and their potential applications [13].

This review addresses recent advances in the devel-

opment of enzymeNP conjugates and their specific

applications for sensing and nanocircuitry design. Itsaim 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 forbiosensing and for the generationof nanostructures

Redox enzymes lack direct electrical contact with elec-

trodes because of the insulation of their active sites by

the protein shell [4]. The electrical wiring of redoxenzymes 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

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: [email protected]

(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 com-

posite materials with new functionalities. The biomoleculeNP 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.

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.

302 FEBS Journal 274 (2007) 302309 2006 The Authors Journal compilation 2006 FEBS


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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 unitswere 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 enzymeNP 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 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.

This paradigm is general and can be applied to other

cofactor-dependent enzymes. For example, the pyrrolo-

quinoline quinone (PQQ)-dependent glucose dehydro-

genase (GDH) was electrically wired by the

reconstitution of apo-GDH on PQQ-functionalizedAuNPs [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-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].

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

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, 18771881. Copyright 2003

AAAS [9].)

I. Willner et al. Nanoparticleenzyme hybrid systems

FEBS Journal 274 (2007) 302309 2006 The Authors Journal compilation 2006 FEBS 303


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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

acts as a reducing agent which reduces AuCl4 and

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

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 biocata-

lyzed oxidation of tyrosine to l-DOPA, a product that

reduced AuCl4

to AuNPs [17]. Similarly, alkalinephosphatase 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),

reduced metal salts (e.g. AuCl4 or Cu2+) and depos-

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.

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 [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 nano-

circuitry and for the preparation of patterned nano-structures. 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 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 200300 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 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, con-

sisting of the substrates specific for the different

enzymes, allows the stepwise, orthogonal formation of

metal nanowires composed of different metals and

A

B

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 fixed time interval of 10 min. (B was adapted with

permission from [15]. Copyright 2005 American Chemical Society.)

Nanoparticleenzyme hybrid systems I. Willner et al.



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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 bythe size of the biocatalytic templates.

The use of coupled enzymeNP 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

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

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 resonanceenergy 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 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

Fig. 3. (A) Dip-pen nanolithographic pattern-

ing of the GOxAuNPs 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.)




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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

peptide sequences specific for different proteases,

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

CdSeZnS QDs. While the tethered dye quenched the

fluorescence of the QD, hydrolytic scission of the dye

and its corresponding removal restored the fluores-

cence.

In a related study [35], the biocatalytic functions

of two enzymes, tyrosinase and thrombin, were

probed by CdSeZnS QDs. A tyrosine-methyl ester-

terminated peptide that included the specific sequence

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 semiconductor NPs not only

yields luminescence probes, but the photogenerated

electronhole pair may also stimulate the generation of

photocurrents. Generation of photocurrents by bio-

moleculeNP conjugates has been demonstrated in sev-

eral 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

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 linkersto the generated catechol ligands.




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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 activa-

tion of the oxidation of lactate by lactate dehydroge-

nase (LDH) proceeds while generating an anodic

photocurrent (Fig. 6A). Photoexcitation of the NPs

resulted in the injection of the conduction-band elec-

trons into the electrode and the concomitant oxidation

of the reduced cytochrome c by the valence-band

holes. The resulting oxidized cytochrome c then medi-

ated 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 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

NO3 to nitrite then enabled the formation of the

cathodic photocurrent. The photocurrents generated

by the biocatalytic cascades at various concentrations

A

B

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 CdSeZnS 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. {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.}

A

B

C

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-

vation of the cytochrome c-mediated reduction of nitrate (NO3) by

nitrate reductase (NR). (C) The photocurrents generated by the bio-

catalytic cascades in the presence of various concentrations of the

substrates (lactatenitrate). (C is taken from [38]; reproduced by

permission of the Royal Society of Chemistry.)




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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 aselectron 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.

Conclusions and perspectives

The unique chemical and physical properties of metal-

lic 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 biomole-

culeNP hybrid systems has recently attracted

immense scientific efforts, and ingenious electronic

and optical biosensors have been assembled, new par-

adigms for the bottom-up construction of nanowires

developed, and highly imaginative nanodevices fabri-

cated. This article has addressed the specific use of

enzymeNP hybrid systems in nanobiotechnology.

The colorimetric assay of the activities of enzymes by

the formation of metallic NPs suggests that new bio-sensor chips and supported biocatalytic optical sen-

sors could be developed in the future. Furthermore,

the size-controlled emission properties of semiconduc-

tor QDs and their use to probe enzyme functions

suggest that, by the application of mixtures of QDs

functionalized with different substrates, the multi-

plexed 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 con-

tinuously being synthesized and developed suggests

that their coupling to biomolecules, specifically

enzymes, will lead to new sensor systems. For exam-

ple, 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 na-

nostructures [39]. Similarly, the coupling of graded

arrays of QDs, which reveal intrastructure energy-

transfer cascades to enzymes, is expected to yield new

functional sensors.

A further application of NPenzyme 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 thesedevelopments have materialized, the use of NPenzyme

hybrids for the biocatalytic synthesis of functional

devices seems feasible. The use of NPbiomolecule

hybrid systems, specifically NPenzyme assemblies, is

in the early phases of development. The results already

obtained promise exciting future developments in this

area of nanobiotechnology.

Acknowledgements

Our research on NPenzyme hybrid systems is suppor-

ted by the IsraeliGerman Program (DIP) and by the

Ministry of Science, Israel.

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Nanoparticle–enzyme hybrid systems