REVIEWARTICLE

Enzymatic biosensors based on the use of metaloxide nanoparticles

Xinhao Shi & Wei Gu & Bingyu Li & Ningning Chen &

Kai Zhao & Yuezhong Xian

Received: 9 May 2013 /Accepted: 16 August 2013 /Published online: 29 August 2013# Springer-Verlag Wien 2013

Abstract Over the past decades, various techniques havebeen developed to obtain materials at a nanoscale level todesign biosensors with high sensitivity, selectivity and effi-ciency. Metal oxide nanoparticles (MONPs) are of particularinterests and have received much attention because of theirunique physical, chemical and catalytic properties. This re-view summarizes the progress made in enzymatic biosensorsbased on the use of MONPs. Synthetic methods, strategies forimmobilization, and the functions of MONPs in enzymaticbiosensing systems are reviewed and discussed. The article issubdivided into sections on enzymatic biosensors based on (a)zinc oxide nanoparticles, (b) titanium oxide nanoparticles, (c)iron oxide nanoparticles, and (d) other metal oxidenanoparticles. While substantial advances have been made inMONPs-based enzymatic biosensors, their applications to realsamples still lie ahead because issues such as reproducibilityand sensor stability have to be solved. The article contains 256references.

Keywords Metal oxide nanoparticles . Enzyme . Biosensor .

Zinc oxide . Titanium oxide . Iron oxide

Introduction

Over the past decades, nanomaterials have attracted extensiveinterests due to their importance in basic scientific researchand potential technological applications. From the point ofview of scientific research, it is well known that the physicaland chemical properties of materials at nanoscale are

obviously different from those of bulk materials because ofthe quantum size effect, small size effect, surface/interfaceeffect and macroscopic quantum tunneling effect.Nanoparticles or nanocrystals, as the fundamental units fornanostructured materials, play very important roles innanoscience. Specifically, metal oxide nanoparticles(MONPs) have attracted much attention for their optical,magnetic and electronic properties.

The fascinating size and shape dependence of physico-chemical properties make MONPs suitable for a wide varietyof applications such as catalysis, organic light emitting diodes,electrochemical energy storage, sensors, etc. It is well knownthat metal oxides have tremendous importance in the field ofheterogeneous catalysis. Debecker andMutin [1] reviewed theprogress of synthesis of MONPs based on non-hydrolytic sol-gel method for catalytical applications. Due to their excep-tional electronic properties, transition metal oxides have beenextensively studied for organic electronics applications.Meyer and coworkers [2] summarized the recent developmentof transition metal oxides such as MoO3, V2O5 and WO3 inorganic light emitting diodes and organic photovoltaic cells.Meanwhile, metal oxide nanostructures are promising elec-trode materials for lithium ion batteries and supercapacitors.Jiang and colleagues [3] reported the advances in metal oxide-based hybrid nanostructure for electrochemical energy stor-age. The emerging nanotechnology opens new exciting op-portunities to explore analytical applications of the nanostruc-tured metal oxide materials. Due to their special shapes,extraordinary chemical and physical properties, metal oxidenanostructures have great promise in the chemical and bio-logical sensing system. It is well known that MONPs not onlyhave high surface area, good biocompatibility and chemicalstability, but also display fast electron transfer ability. All thesefeatures make MONPs ideal immobilization matrices as wellas transduction platform and/or mediators. For example,Shaidarova [4, 5] conducted a series of researches on

X. Shi :W. Gu :B. Li :N. Chen :K. Zhao :Y. Xian (*)Department of Chemistry, East China Normal University,Shanghai 200062, Chinae-mail: yzxian@chem.ecnu.edu.cn

Microchim Acta (2014) 181:1–22DOI 10.1007/s00604-013-1069-5

chemically modified electrodes using metal oxides as modi-fiers. The results indicate that metal oxides can improve thesensitivity the electrode obviously.

Nanofabricated metal oxides also have been successfullyapplied in the field of biosensing system. Recently, some re-views concentrated on nanostructured metal oxides based bio-sensors were published. For example, Haun [6] reviewed theuse of magnetic nanoparticles for detection of biomolecules andcells based on magnetic resonance effects. Xu and Wang [7]summarized the applications of magnetic micro/nano particlesin the development of immuno-, enzyme, DNA and aptamerelectrochemical biosensors. The application of nanostructuredZnO in the field of biosensing system also was reviewed [8, 9].Very recently, Solanki and coworkers [10] demonstrated theprogress of application of nanostructured metal oxide (NMO)in biosensors. They provided a comprehensive comment onenzymes, nucleic acids, antibodies and whole cell-based NMObiosensing system. However, until now, a review focus onenzymatic biosensors based on MONPs has not been reported.It is well known that enzymes and enzyme mimetics can offergreat amplification power through efficient catalytic turnover ofsubstrates. The combination of enzymes with MONPs couldprovide a versatile platform for biosensing with various signalreadout strategies, such as electrochemical, colorimetric, fluo-rimetric, chemiluminescence and so on. In this paper, we aim torepresent a comprehensive and critical review of the progressand future perspectives of MONPs based enzymatic biosensingsystem. The review is structured in accordancewith the kinds ofMONPs and is subdivided into sections on (a) zinc oxidenanoparticle-based enzymatic biosensors, (b) titanium oxidenanoparticle-based enzymatic biosensors; (c) iron oxidenanoparticle-based enzymatic biosensors, and (d) on other met-al oxide nanoparticle-based enzymatic biosensors. It should bepointed out that, in some MONPs based biosensing systems,the recognition mechanisms is not based on the direct reactionbetween enzyme and analyte, for example, iron oxidenanoparticles as enzyme mimetics and some examples ofimmunobiosensors. In order to make more comprehensiveunderstanding the roles of MNOPs in enzymatic biosensingsystem, above-mentioned contents were also involved in thisreview.

Zinc oxide nanoparticle based enzymatic biosensors

As a semiconductor material, nanostructured ZnO has re-ceived broad attention. Tremendous efforts have been madeto synthesize nanostructured ZnO, including solid–vapourphase thermal sublimation technique and wet-chemical syn-thesis. Recently, various strategies exploited to grow ZnOnanostructures were reviewed and these controllable syntheticmethods enabled the versatile applications in the fields of solarcells [11], sensors [12], photocatalysis [13], and luminescent

materials [14], etc. Figure 1 shows the typical TEM images ofZnO nanoparticles synthesized with controlled sizes and mor-phologies using [Zn(c-C6H11)2] as precursor at room-temperature [15].

As a biomimetic material, ZnO provides a versatile plat-form for biomolecules loading [16]. ZnO nanoparticles withhigh isoelectric point are suitable for the adsorption of low-isoelectric point enzymes for biosensor fabrication (Table 1).

A mediator-free Tyr biosensor based on ZnO nanoparticleswas developed by Li for the determination of phenolic com-pounds [31]. In this work, ZnO nanoparticles prepared by ahydrothermal method were dispersed in the chitosan solutionto form ZnO/chitosan matrix, and then Tyr was adsorbed onZnO nanoparticles by electrostatic interactions for phenolbiosensing. In addition, the direct electrochemistry of hemo-globin (Hb) [44], microperoxidase [45], GOx [17, 46], andHRP [28] were observed at ZnO nanoparticles modified elec-trode. In order to improve the conductivity and enhance theelectron transfer, Au nanoparticles [29, 30], reduced grapheneoxide [18] and MWCNTs [19] were modified on the surfaceof electrode with ZnO nanoparticles and the direct electrontransfer between the enzyme and electrode were realized.

Due to its simplicity and free pretreatment, electrochemicaltechniques have gainmuch interest for growing ZnO nanostruc-tures. Ahmad et al. [47] fabricated a cholesterol biosensor basedon Pt-ZnO nanomaterials, in which ZnO nanospheres wereelectrochemical deposited on the glassy carbon (GC) electrode.As shown in Fig. 2, the morphology characterizations demon-strate ZnO nanospheres with diameter over the range of 10–200 nm. The doped Pt nanoparticles assembled on the surfaceof the first spherical agglomerate and finally the nanocompos-ites display a fullerene-like spherical shape with diameters ofabout 50–200 nm. Functionalized with ChOx by physicaladsorption, the biosensor exhibits a very high sensitivity of1886.4 mA M−1 cm−2 to cholesterol with a response time lessthan 5 s. Deshpande [48] developed a potentiometric glucosebiosensor using core-shell nanocomposite based on zinc oxideencapsulated chitosan-graft-poly(vinyl alcohol) (ZnO/CHIT-g-PVAL). In this system, ZnO/CHIT-g-PVALwas synthesized bywet-chemical method. The nanocomposite was coated on thesurface of indium-tin oxide (ITO) glass substrate and GOx wasfurther immobilized through the electrostatic interaction. Theobtained biosensor shows a linear potential response to theglucose concentration ranging from 2.0 μM to 1.2 mM. Aphotoelectrochemical system based on GOx/ZnO nanoparticleswas described by Ren [49]. The experimental results show thatthe photovoltaic effect of the ZnO nanoparticles could enhancethe catalytic activity of GOx and improve the sensitivity ofglucose biosensor. The detection limits of enzyme electrodecontaining ZnO nanoparticles in the dark and under illumina-tion were 5.6×10−6 M and 1.1×10−4 M, respectively.

Recently, Kim developed a fluorescence glucose bio-sensor based on the collisional quenching mechanism

2 X. Shi et al.

(Fig. 3) [50]. ZnO nanocrystals were synthesized usingmercaptoundecanoic acid (MUA) as capping agent andbiolinker. Through covalent modification, GOx wasimmobilized onto ZnO nanocrystals. The resulting ZnO-MUA-GOx bioconjugate shows a linear decrease in thephotoluminescence (PL) intensity due to the collisionalquenching by H2O2 generated from enzymatic reaction overthe range of 1.6 to 33.3 mM.

Titanium oxide nanoparticle based enzymatic biosensors

TiO2 is one of the most prominent materials in various appli-cations related to catalysis, photovoltaic devices, sensors andpaintings. Over the past decades, tremendous efforts for syn-thesis, modifications and applications of TiO2 nanomaterialshave been conducted and a series of reviews have demonstrat-ed the breakthroughs in these fields. Since Cosnier and co-workers [51] reported an amperometric glucose biosensorbased on mesoporous TiO2 films in 1997, the applica-tions of nanostructured TiO2 for enzyme immobilizationand biosensor design have attracted considerable atten-tion. Through physical adsorption or covalent immobili-zation, different enzymes have been modified onto thesurface of TiO2 nanoparticles [52–58]. The detail infor-mation for these enzymatic biosensors is listed inTable 2.

It has been reported that the morphology and structure ofTiO2 nanomaterials have important effects on the performance

of biosensor. Li [74] reported that size-controllable and well-dispersed TiO2 nanodots could be directly fabricated on ITOelectrodes through a facile PSIA method. The sensitivity ofthe biosensors was significantly increased by adjusting theaverage size of the TiO2 nanodots from 30 to 79 nm. Xie [93]demonstrated that TiO2 nanoparticles with different morphol-ogies and sizes could be achieved by controlling the synthesisconditions, such as reaction time, solution pH, temperatureand growth precursors. Figure 4 shows different morphologiesof TiO2 nanospheres in Xie’s work, in which TiO2

nanoparticles were prepared within different hydrothermalreaction time. The TiO2 nanosphere shows a pretty core-shell structure and the size is successfully controlled varyingwith time. After coated HRP and TiO2 onto GC electrode withNafion, the direct chemistry of HRP was observed.

TiO2 is very famous for its unique photocatalytic ability,which is favorable for photoelectrochemical applications.Li [94] reported construction of a competitive photo-electrochemical immunosensor for alpha-fetoprotein (AFP) de-tection using TiO2 nanoparticles to enhancement the detectionsensitivity. Figure 5 shows the fabrication process of thephotoelectrochemical immunosensor and correspondingelectron-transfer mechanism.

Iron oxide nanoparticle based enzymatic biosensors

Well-established magnetic nanoparticles with appropriate sur-face chemistry have been widely used in pollution

Fig. 1 TEMmicrographs of ZnOnanoparticles. a ZnO nanorodsgrown under standard conditions;b ZnO nanodisks following aslow oxidation/evaporationprocess in THF (2 weeks); c ZnOnanodisks using dodecylamine(DDA) instead ofhexadecylamine (HDA) as thestabilizing ligand under standardconditions; d ZnO nanodisksusing octylamine (OA) instead ofHDA under standard conditions[ref. 15]

Enzymatic biosensors 3

remediation [95], chemical or biological separation [96],bioimaging [97, 98], targeted drug delivery [99–101], diseasesdiagnostics and therapy [102, 103], enzyme immobilization[104], biosensing [105, 106], etc. Among them, iron oxidenanoparticles, such as Fe3O4 and γ-Fe2O3, are the most prom-ising candidates. Controlling the size, size distribution, shape,crystal structure, defect distribution and surface structure ofiron oxide nanoparticles is very important for their applica-tions. Over the past decades, numerous synthetic approacheshave been developed for synthesis of iron oxide nanoparticles.The advances in the synthesis, protection as well as surfacefunctionalization were reviewed by different authors. Figure 6shows an example of controlled synthesis iron oxidenanoparticles in reference [107].

Due to their superior biocompatibility and large surfacearea, iron oxide nanoparticles can be used as nanocarrier forenzyme. GOx is the most widely used enzyme in the field ofenzyme biosensor. Other enzymes, such as hydrolase, HRP,Mb, Hb, creatinase, Lac, lactate dehydrogenase (LDH) andnicotinamide adenine dinucleotide (NAD+), etc. were success-fully immobilized on the surface of iron oxide nanoparticlesvia physical adsorption or covalent immobilization. Differentkinds of biosensors based on electrochemical and opticalmethods were developed for application in various fields,which were listed in Tables 3 and 4, respectively.

In 2004, an enzyme biosensor based on iron oxidenanoparticles was reported by Rossi [176]. In this work,GOx-Fe3O4 nanoparticle bioconjugate was synthesized by

Table 1 ZnO nanoparticles for enzyme immobilization and the performance of enzymatic biosensors

Analyte Enzyme Matrix Immobilizationmethod

Analyticalmethod

Performances Reference

Analytical range Limit ofdetection

Glucose GOx porous ZnO Electrostatic Amperometric 0.05×10−5–8.2×10−3 M 1×10−5 M [17]

Glucose GOx ZnO/RGO Electrostatic Amperometric 2×10−5–6.24×10−3 M 2×10−5 M [18]

Glucose GOx ZnO/CNTs Adsorption Amperometric 6.67×10−6–1.29×10−3 M 2.22×10−6 M [19]

Glucose GOx ZnO Crosslinking Amperometric 0–4×10−3 M 2×10−6 M [20]

Glucose GOx ZnO/Cu Electrostatic Amperometric 1×10−3–1.5×10−2 M − [21]

Glucose GOx ZnO/CNTs Electrostatic Amperometric 1×10−4–1.6×10−2 M 2.50×10−7 M [22]

Glucose GOx hollow ZnO Electrostatic Amperometric 5.0×10−6–1.315×10−2 M 1.0×10−6 M [23]

Cholesterol ChOx ZnO Adsorption Amperometric 1×10−9–5×10−7 M 0.37±0.02×10−9 M [24]

Cholesterol ChOx Pt/Au/ZnO Electrostatic Amperometric 1×10−7–7.593×10−4 M 3×10−8 M [25]

Cholesterol ChOx ZnO Electrostatic Amperometric 1.2×10−2–1.293×10−2 M − [26]

Cholesterol ChOx ZnO Electrostatic Amperometric 25–400 mg·dL−1 − [27]

H2O2 HRP ZnO Electrostatic Amperometric 2×10−5–3.5×10−4 M [28]

H2O2 HRP ZnO/Au Electrostatic Amperometric 1.5×10−5–1.1×10−3 M 9.0×10−6 M [29]

H2O2 HRP ZnO/Au Covalent Amperometric 1.5×10−6–4.5×10−4 M 7.0×10−7 M [30]

Phenol Tyr ZnO Electrostatic Amperometric 1.5×10−7–6.5×10−5 M 5.0×10−8 M [31]

Phenol Tyr ZnO Electrostatic Amperometric 1.5×10−7–4.0×10−5 M 8.0×10−8 M [32]

Urea Urease ZnO Electrostatic Amperometric 1×10—3–1×10−1 M − [33]

Urea Urease ZnO Electrostatic Amperometric − 13.5 mg·dL−1 [34]

Uric acid Uricase ZnO Electrostatic Amperometric 5.0×10−6–1.0×10−3 M 2.0×10−6 M [35]

Uric acid Uricase ZnO/CNTs Electrostatic Amperometric 5.0×10−6–1×10−3 M − [36]

Uric acid Uricase ZnO Adsorption Amperometric 1×10−4–5.9×10−4 M 2.56×10−6 M [37]

Xanthine XOD ZnO/CNTs/polyaniline

Electrostatic Amperometric 1×10−7–1×10−4 M 1×10−7 M [38]

Xanthine XOD ZnO/polypyrrole Electrostatic Amperometric 8×10−7–4×10−5 M 8×10−7 M [39]

Acetylcholineand choline

ChOx ZnO Adsorption Amperometric Ach: 1.0×10−6–1.5×10−3 MCh: 0–1.6×10−3 M

Ach: 6.0×10−7 MCh: 5.0×10−7 M

[40]

L-lactate LOD ZnO/CNTs Electrostatic Amperometric 2×10−4–2×10−3 M 6×10−6 M [41]

Pesticide AChE ZnO Electrostatic Amperometric 2.5×10−7–1.5×10−6 and1.75×10−6–1×10−5 M

1×10−8 M [42]

Creatinine Creatinine amidohydrolase,creatine amidino-hydrolase and SAO

ZnO/CNTs/polyaniline

Electrostatic Amperometric 1×10−5–6.50×10−4 M 5×10−7 M [43]

GOx glucose oxidase; ChOx choline oxidase; HRP horseradish peroxidase; Tyr Tyrosinase; XOD xanthine oxidase; LOD lactate oxidase; AChEacetylcholinesterase

4 X. Shi et al.

covalent immobilizing GOx on amino-modified magneticnanoparticles. The experimental results show that

functionalization of the magnetic nanoparticle surface withamino groups greatly increases the amount and activity of

Fig. 2 a a) SEM image of ZnOnanospheres. b) TEM(transmission electronmicroscopy) image along withEDS (energy dispersive X-rayspectroscopy) of ZnOnanospheres. (c) HRTEM (high-resolution transmission electronmicroscopy) image of individualZnO nanosphere. (d–f) TypicalSEM images of the as-depositedPt-incorporated ZnOnanospheres. (d) Low-magnification image of sphericalagglomerates of nanospheres. (e)Individual agglomerate spheretaken from the arrow point in (d).(f) Pt−ZnO nanospheres on thesurface of self-assembledmicrospheres from the arrowpoint in (e); b Cyclicvoltammetric sweep curve of theNafion/ChOx/PtZONS/GCEelectrode in the absence (CVa)and in the presence (CV c) of100 μM cholesterol in PBsolution (pH 6.8) at a scan rate of50 mV·s−1; CV curve of theNafion/ChOx/ZnO/GCEelectrode in the presence of100 μM cholesterol (CV b) in PBsolution (pH 6.8) at scan rate of50 mV·s−1; c Amperometricresponse of the biosensor basedon ZnO and Pt−ZnO nanospheresto different cholesterolconcentrations at +0.2 V in stirredpH 6.8 PB solutions [ref. 47]

Fig. 3 a Variation in PL spectra with H2O2 concentration, and b schematic presenting a collisional quenching mechanism causing decrease in PLintensity of ZnO nanocrystals [ref. 50]

Enzymatic biosensors 5

the enzyme. Using Ru(phen)3 as oxygen-sensitive fluores-cence probe, the GOx conjugated magnetic nanoparticles

could be used as a glucose sensors. In Margo’s work [177],γ-Fe2O3 nanoparticles (20–40 nm) with well-defined

Table 2 Enzymatic biosensors based on TiO2 nanoparticles

Analyte Enzyme Matrix Immobilizationmethod

Analytical method Performances Reference

Analytical range Limit ofdetection

Glucose GOx TiO2/CNTs Covalent Amperometric 1.8×10−6–2.66×10−4 M 4.4×10−7 M [59]

Glucose GOx TiO2/G Adsorption Amperometric 0–8×10−3 M – [60]

Glucose GOx TiO2/Au/CNTs

Electrostatic Amperometric 6×10−6–1.2×10−3 M 1×10−7 M [61]

Glucose GOx TiO2 Adsorption Amperometric 0–3×10−3 M − [62]

Glucose GOx TiO2 Covalent Amperometric 1.53×10−4–1–1.3×10−3 M 5.1×10−5 M [63]

Glucose GOx TiO2/SiO2 Adsorption Phosphorescent 1.0×10−9–1.0×10−2 M 1.2×10−10 M [64]

Glucose GOx TiO2/Ru Crosslinking Potentiometric 100–500 mg·−1 – [65]

Glucose GOx Cu/TiO2 Adsorption Amperometric 5×10−7–3×10−3 M – [66]

Glucose GOx TiO2/Au − Electrochemiluminescence 5.0×10−7–4.0×10−3 M 2.5×10−7 M [67]

Glucose GOx TiO2/PVA-g-PVP

Entrapment Amperometric 0–9×10−3 M − [68]

Glucose GOx TiO2 Entrapment Amperometric 5×10−4–1×10−3 M − [69]

Glucose,glutamateand urea

Urease, glucosedehydrogenaseand GDH

TiO2 Entrapment Fluorimetric Urea: 1×10−5–8×10−3 MGlucose: 4×10−5–1×

10−2 MGlutamate: 1×10−5–1×

10−2 M

Urea: 3.1×10−6 MGlucose: 5.4×

10−6 MGlutamate: 7.8×

10−6 M

[70]

H2O2 HRP TiO2 Entrapment Amperometric 7.5×10−6–1.23×10−4 M 2.5×10−6 M [71]

H2O2 HRP TiO2/Au/CNTs

Adsorption Amperometric 2.3×10−6–2.4×10−3 M 7.0×10−7 M [72]

H2O2 HRP TiO2/Au Adsorption Amperometric 4.1×10−5–6.3×10−4 M 5.9×10−6 M [73]

H2O2 HRP TiO2

nanodotsAdsorption Amperometric 1×10−6–7.8×10−4 M 8.5×10−7 M [74]

H2O2 HRP TiO2/PVA/CNTs

Entrapment Amperometric 5×10−7–2.7×10−6 M 1×10−8 M [75]

H2O2 HRP TiO2 Entrapment Amperometric 4.0×10−6–1.0×10−3 M 8.0×10−7 M [76]

H2O2 Mb and HRP TiO2 Electrostatic Amperometric 1×10−6–1.6×10−4 M 5×10−7 M [77]

H2O2 Hb TiO2/Au Covalent Amperometric 1.4×10−6–1.6×10−3 M 3.7×10−7 M [78]

Catechol Tyr TiO2 Adsorption Electrochemiluminescence 1.25×10−7–1.625×10−4 M 4.17×10−8 M [79]

Catechol Tyr TiO2 Adsorption Amperometric Up to 1.4×10−4 M 5×10−7 M [80]

Phenol Tyr TiO2/CNTs Crosslinking Amperometric 1×10−7–5×10−5 M 9.5×10−8 M [81]

Phenols Tyr TiO2 Entrapment Amperometric 7.5×10−8–6×10−6 M 1×10−8 M [82]

Glutamate GDH TiO2 Entrapment Fluorimetric 4×10−5–1×10−2 M 5.5×10−6 M [83]

Humanchorionicgonadotropin

HRP TiO2-Au/PB/CNTs

Covalent Amperometric 0.05 to 150 mIU·mL−1 0.023 mIU·mL−1 [84]

Lactic acid LDH TiO2 Entrapment Amperometric 1×10−6–2×10−5 M 4×10−7 M [85]

Nitrophenol HRP TiO2/Au Covalent Amperometric 3×10−7–1.2×10−4 M 9×10−8 M [86]

Organophosphate AChE TiO2/G Adsorption Amperometric 0.001–0.015 and 0.015–2ug·mL−1

0.3 ng·mL−1 [87]

2,4-Dichlorophe-nol

Lac TiO2 Crosslinking Thermometric 5×10−4–1×10−3 M 5×10−5 M [88]

AFP HRP TiO2/Au Covalent Amperometric − 29 pg·mL−1 [89]

Progastrinreleasingpeptide

GOx TiO2/Au Covalent Amperometric 10.0–500 pg·mL−1 3.0 pg·mL−1 [90]

Superoxide anion XOD CMC/G/TiO2 Crosslinking Amperometric 1.5×10−9–2×10−3 M 1.25×10−6 M [91]

Trichlorfon AChE PbO2/TiO2 Adsorption Amperometric 1×10−8–2×10−5 M 1×10−10 M [92]

Mb myoglobin; GDH glutamate dehydrogenase; Lac laccase

6 X. Shi et al.

stoichiometric structure were synthesized. The existence ofOH− groups at γ-Fe2O3 nanoparticles could act as chargebarriers preventing the aggregation of nanoparticles and en-abling a reversible binding with positive charged organicsubstances, such as Rhodamine B isothiocyanate. Afterimmobilized with GOx, the fluorimetric biosensor shows arate constant of 32.7 s–1 toward glucose oxidation. Shi [183]reported using circular strand-displacement amplificationchemiluminescence for point mutation detection at room tem-perature. Based on the circular strand-displacement amplifi-cation and HRP-catalyzed luminol–PIP–H2O2 reaction, thesensitivity was improved obviously. In addition, the magneticseparation was used to reduce the background signal in het-erozygous state and T4 DNA ligase to produce good selectiv-ity. Besides above-mentioned examples, some other opticalprobes based on enzyme and iron oxide nanoparticles werealso reported to measure different analytes, such as biogenicamines [178], enzyme inhibition assays [184], H2O2 andglucose [179].

Electrochemical method is the universal strategy in enzymebiosensor, such as potential [167] and capacitive [163] mea-surements. The most widely used electrochemical method isamperometry. For instance, amperometric Tyr biosensor basedon Fe3O4 nanoparticles-chitosan nanocomposite was devel-oped for the detection of phenolic compounds [140]. The largesurface area of Fe3O4 nanoparticles and the porous morphol-ogy of chitosan lead to a high loading of enzyme. Based on

enzyme amplification and magnetic enrichment and separa-tion, enzyme biosensors based on iron oxide have been widelyused in immunoassay. A three-layer composite composed ofFe3O4 magnetic core, prussian blue interlayer and gold shellwas used to fabricate an electrochemical immunosensor byfunctionalized with bienzyme of HRP and GOx (Fig. 7). Dueto the signal amplification from the nanobioconjugates, anelectrochemical immunosensing system was developed usingcarcinoembryonic antigen (CEA) and AFP as model. With theaddition of glucose, the immunosensor shows linear ranges of0.04–80.0 ng·mL−1for CEA and 0.074–142.0 ng·mL−1forAFP [137].

In order to improve the sensitivity of the enzymatic bio-sensors, some electron mediators or electron promoters wereintroduced into the biosensing system. For example, an am-perometric glucose biosensor was developed by entrappingGOx in chitosan composite doped with ferrocene monocar-boxylic acid-modified Fe3O4@SiO2 nanoparticles [120].With the aid of a permanent magnet, the magneticbionanoparticles were attached to the surface of CPE andacted as mediator to transfer electrons between the enzymeand the electrode. Eguílaz [145] developed a bienzymebiosensing system for the determination of cholesterol, inwhich Fe3O4 nanoparticles functionalized with glutaralde-hyde and poly(diallyldimethylammonium chloride) coatedMWCNTs were used as platforms for immobilization ofChOx and HRP. Using hydroquinone as a redox mediator,

Fig. 4 a SEM and TEM (insets) images of the calcined TiO2 micro-spheres prepared with different hydrothermal reaction times: (a) 1 h, (b)3 h, (c) 6 h , (d) 12 h , (e) 48 h, and (f) 96 h; b Cyclic voltammograms ofthe TiO2–48/Nafion/GC electrode (curve a), HRP/Nafion/GC electrode

(curve b), HRP/TiO2–1/Nafion/GC electrode (curve c), HRP/TiO2–6/Nafion/GC electrode (curve d) and HRP/TiO2–48/Nafion/GC electrode(curve e) in 0.1 M pH 7.5 PBS [ref. 93]

Enzymatic biosensors 7

Fig. 5 a Fabrication process of photoelectrochemical immunosensor andb corresponding electron-transfer mechanism c Photocurrent responsesand d corresponding calibration curve of ITO/TiO2/CS/anti-AFP/BSAelectrode in the presence of mixture containing 10 μL of AFP–CdTe–

GOx bioconjugates and 10 μL of different concentrations of AFP, whichwere measured in 0.1 M phosphate buffer solution (pH 7.4) containing60 mM glucose [ref. 94]

8 X. Shi et al.

the bienzyme biosensing system provided a sensitive andselective strategy for cholesterol measurement at a low detec-tion potential of −0.05 V.

Due to the supermagnetism of iron oxide nanoparticles, themagnetoswitchable biosensing system was developed. Tyrfunctionalizedmagnetic Fe3O4 nanoparticles were grafted ontoMWCNTs and the obtained MNP/Tyr/MWCNTs nanocom-positeswere utilized to design amagnetoswitchable biosensingsystem by magnetically loaded the nanocomposites on theworking electrode [141]. It allows an on-off bioelectrocatalyticswitching that controls redox reactions at the electrode surfacethrough the introduction, removal, and relocation of the exter-nal magnet (Fig. 8). In our research group, themagnetoswitchable bioelectrocatalytic systems were also de-veloped [185, 186]. A simple and versatile method for theintroduction of redox mediator (ferrocene) onto the surface ofmagnetic nanoparticles was developed based on “click” chem-istry. Due to the magnetism of magnetic nanoparticles and theelectrocatalytic activity of ferrocene unites, a recyclable,magneto-switchable bioelectrocatalytic system for glucose ox-idation in the presence of GOx was developed by simplealternate positioning of an external magnet.

In 2007, Gao [122] reported that Fe3O4 nanoparticles havean intrinsic peroxidase-like activity for the first time. Based onthe magnetism and peroxidase activity of Fe3O4 nanoparticles,they developed a novel capture-detection immunoassay forcardiac troponin I. After that, the enzyme mimetic activity ofiron oxide nanoparticles was used to design differentbiosensing systems. Wei [187] reported a colorimetric assaystrategy for glucose using Fe3O4 nanoparticles as peroxidasemimetics. Colorimetric method based on peroxidase activityof Fe3O4 nanoparticles was also used to detection thrombin[169], H2O2 [170], ethanol and methanol [171], galactose[172], organophosphorus pesticides and nerve agents [188],and so on. In addition, the peroxidase mimetic activity ofFe3O4 nanoparticles was also used in electrochemicalbiosensing system for H2O2 and glucose [121, 133, 134, 189].

Other metal oxide nanoparticle based enzymaticbiosensors

The above-mentioned MONPs are the most widely used ox-ides in enzyme-based biosensors. Relatively, the examples of

enzymatic biosensors based on other metal oxides are rare.However, the inherent advantages of metal oxide such as largesurface area, excellent biocompatibility, nontoxicity and cata-lytic ability, make it possible for enzyme immobilization andbiosensor development. For example, zirconia nanoparticlesand their nanocomposites have been reported to be used forenzyme immobilization, such as HRP [190–192], GOx [193,194], AChE [195], Hb and Bilirubin oxidase (BOD). Anelectrochemical quartz crystal microbalance (EQCM) immu-noassay was developed utilizing ZrO2 nanoparticles for selec-tive capture phosphorylated AChE (Phospho-AChE) due totheir strong affinity for phosphoric groups (Fig. 9) [196].HRP-labeled anti-AChE antibodies were employed to recog-nize the captured phosphorylated proteins. This immunoassaycould be used to measure Phospho-AChE in human plasmawith a detection limit of 0.020 nM.

Nickel oxide also is a promising material for enzymeimmobilization owing to its high biocompatibility and largesurface area. GOx, catalase and Tyr were reported to beimmobilized onto electrodeposited nickel oxide nanoparticlesand the direct electron transfer of enzyme was realized [197].In addition, the direct electron transfer of catalase on nano-sized NiO/MWCNTs composite film was also reported [198].Gupta’s research group [199] reported that uricase was phys-ically adsorpted on NiO thin film prepared by radio frequency(RF) sputtering technique. The biosensor exhibits efficientsensing performance towards uric acid due to the good elec-tron transport property and nanoporous morphology.Moreover, the RF sputtered NiO thin film was also used toimmobilize GOx for glucose biosensing due to its high holemobility [200]. Recently, ChOx, XOD, cytochrome c (Cyt c),Urease and alcohol dehydrogenase enzyme were reported tobe physisorbed on NiO nanoparticles or nanocomposite todevelop enzymatic biosensor [201–205].

Although MnO2 bulk material is a poor catalyst, MnO2

nanomaterials show high catalytic ability. Enzyme field-effecttransistors (ENFET) based on the catalytic or special reactionactivity of MnO2 nanoparticles were reported by immobiliza-tion of enzyme and MnO2 at the gate surface [206]. Forinstance, Luo [207] developed a glucose-sensitive ENFETbased on MnO2 nanoparticles. In this system, the ENFETworked with local pH change in biomembranes, whichresulted from the formation of gluconic acid by combingGOxwithMnO2. Amperometric glucose biosensors were also

Fig. 6 Schematic illustration ofthe formation of Fe3O4

nanocrystals. The middle andright panels are TEM images ofthe as-synthesized nanocrystalstaken at different reaction times.[ref. 107]

Enzymatic biosensors 9

Table 3 Electrochemical enzyme biosensors based on iron oxide nanoparticles

Analyte Enzyme Matrix ImmobilizationMethod

Analyticalmethod

Performances Reference

Analytical range Limit of detection

Glucose GOx Pt/ FexOy /MWCNTs

Adsorption Amperometric 6.0×10−6–6.2×10−3 M 2.0×10−6 M [108]

Glucose GOx Cobalt ferrite Electrostatic Amperometric − − [109]

Glucose GOx Fe3O4/SiO2 Covalent Amperometric 2.5×10−4–2.0×10−3 M [110]

Glucose GOx Fe3O4/SiO2/CNTs

Adsorption Amperometric 1×10−4–3×10−2 M 8×10−7 M [111]

Glucose GOx Au/Fe3O4 Covalent Amperometric 5×10−5–2×10−2 M − [112]

Glucose GOx γ-Fe2O3/C Adsorption Amperometric 2×10−4–1×10−2 M 8×10−5 M [113]

Glucose GOx Au/Fe3O4 Entrapment Amperometric 2.0×10−6–2.6×10−3 M 3.3×10−7 M [114]

Glucose GOx CoFe2O4/Au Adsorption Amperometric 0.01–0.1 M − [115]

Glucose GOx Fe3O4 Adsorption Amperometric 3.97×10−3 M [116]

Glucose GOx Fe3O4 Entrapment Amperometric 2×10−5–1.875×10−3 M 6.5×10−6 M [117]

Glucose GOx and HRP Fe3O4 Crosslinking Amperometric 1×10−3–8×10−3 M 1×10−5 M [118]

Glucose GOx Fe3O4/PB Covalent Amperometric 5.0×10−7–8.0×10−5 M 1.44×10−5 M [119]

Glucose GOx Fe3O4/SiO2 Entrapment Amperometric 1×10−5–4×10−3 M 3.2×10−3 M [120]

Glucose GOx Fe3O4 Crosslinking Amperometric 6×10−6–2.2×10−3 M 6×10−6 M [121]

Glucose GOx Fe3O4 Crosslinking Amperometric 5×10−4–1×10−2 M 2×10−4 M [122]

H2O2 HRP Fe3O4 Adsorption Amperometric 2.0×10−4–1.2×10−2 M 1.0×10−4 M [123]

H2O2 HRP NiFe2O4 Adsorption Amperometric 3×10−4–1.2×10−3 M − [124]

H2O2 HRP Fe3O4 Adsorption Amperometric 2×10−7–6.8×10−4 M 7.8×10−8 M [125]

H2O2 HRP Fe2O3 Adsorption Amperometric 4×10−7–3×10−4 M 5×10−8 M [126]

H2O2 HRP, Hb and Mb Fe3O4/Al2O3 Adsorption Amperometric Hb: 0.51×10−7–1.112×10−4 MMb: 4.3×10−7–1.158×10−4 MHRP: 8.5×10−7–1.015×10−4 M

Hb: 1.6×10−7 MMb: 1.4×10−7 MHRP: 2.8×10−7 M

[127]

H2O2 Mb Au/Fe3O4 Adsorption Amperometric 1.28×10−3–0.283 M 4×10−4 M [128]

H2O2 Mb Fe3O4 Crosslinking Amperometric 6.9×10−5–2.9×10−4 M and 2.9×10−4–3.0×10−3 M

2.1×10−5 M [129]

H2O2 Hb Fe3O4/SiO2 Covalent Amperometric 2.03×10–6–4.05×10–3 M 3.2×10−7 M [130]

H2O2 HRP Fe3O4/SiO2 Electrostatic Amperometric − 4.3×10−7 M [131]

H2O2 andglucose

HRP Fe3O4/GO Covalent Amperometric H2O2: 4×10−4–4×10−2 M

Glucose: 4×10−3–5.6×10−2 M− [132]

H2O2 peroxidase mimic Fe3O4 − Amperometric 2×10−4–2×10−3 M 1×10−5 M [133]

H2O2 peroxidase mimic Fe3O4 − Amperometric 4.18×10−6–8×10−4 M 1.4×10−6 M [134]

AFP HRP Au/Fe3O4 Covalent Amperometric 0.01–200 ng·mL−1 5 pg·mL−1 [135]

AFP α-AFP antibody Fe2O3 Covalent Amperometric 1–80 ng·mL−1 0.5 ng·mL−1 [136]

CEA and AFP GOx and HRP Au/PB/Fe3O4 Covalent Amperometric CEA: 0.04–80.0 ng·mL−1

AFP: 0.074–142.0 ng·mL−1CEA: 4 pg·mL−1

AFP: 0.014 to142.0 ng·mL−1

[137]

Catechol Lac Fe3O4/SiO2 Covalent Amperometric 7.5×10−7–2.75×10−4 M 7.5×10−7 M [138]

Catechol Lac Fe3O4 Covalent Amperometric 7.5× 10−7–4.4× 10−4 M − [139]

Catechol Tyr Fe3O4 Adsorption Amperometric 8.3×10−8–7.0×10−5 M 2.5×10−8 M [140]

Catechol Tyr Fe3O4/CNTs Covalent Amperometric 1×10−5–1.20×10−4 M 7.61×10−6 M [141]

Phenol Tyr MgFe2O4/SiO2

Covalent Amperometric 1×10−6–2.5×10−4 M 6.0×10−7 M [142]

Phenol Tyr Fe3O4 Crosslinking Amperometric 1.0×10−9–1.0× 10−5 M 1×10−9 M [143]

Phenol HRP Fe3O4 Covalent Amperometric − − [144]

Cholesterol HRP and ChOx Fe3O4 Crosslinking Amperometric 1×10−5–9.5×10−4 M 8.5×10−6 M [145]

Clozapine HRP SiO2/ Fe3O4-γ-Fe2O3

Covalent Amperometric 5×10−6–2.5×10−5 M − [146]

Creatine Creatinase Fe3O4 Adsorption Amperometric 2×10−7–3.8×10−6 M 2×10−7 M [147]

DNA HRP Fe3O4/Au Electrostatic Amperometric 5.0×10−11–5×10−7 M 7.1×10−12 M [148]

Dopaquinone Tyr γ-Fe2O3 Covalent Amperometric − − [149]

Escherichia coli HRP Au/Fe2O3 Adsorption Amperometric 1×103–5×105 cfu·mL−1 5 cfu·mL−1 [150]

Ethinylestradiol HRP Fe2O3 Covalent Amperometric 0.1–1,500 ng·L−1 – [151]

10 X. Shi et al.

Table 3 (continued)

Analyte Enzyme Matrix ImmobilizationMethod

Analyticalmethod

Performances Reference

Analytical range Limit of detection

Fructosyl valine Fructosyl aminoacid oxidase

Fe3O4 Covalent Amperometric 0–2×10−3 M 1×10−4 M [152]

Hydroquinone Lac Fe3O4/SiO2 Covalent Amperometric 1×10−7–1.375×10−4 M 1.5×10−8 M [153]

Methyl parathion Hydrolase Au/Fe3O4 Electrostatic Amperometric 0.5–1,000 ng·mL−1 0.1 ng·mL−1 [154]

Mycobacteriumtuberculosis

Thermophilichelicase

Au/Fe2O3 − Amperometric 0.01–10 ng·μL−1 0.01 ng·μL−1 [155]

NADH LDH and NAD+ Fe3O4/CNTs Covalent Amperometric 5×10−5–5×10−4 M 5×10−6 M [156]

Ochratoxin A HRP γ-Fe2O3/Au Covalent Amperometric 0.78–8.74 ng·mL−1 0.07±0.01 ng·mL−1

[157]

Ochratoxin A HRP γ-Fe2O3 Covalent Amperometric 0.027–0.033 ng·mL−1 0.11±0.01 ng·mL−1

[158]

Organophos-phoruspesticides

AChE Fe3O4 – Amperometric – 4.0× 10−13 M [159]

Organophos-phorusesticides

AChE Au/Fe2O3 Covalent Amperometric 1.0×10−3–10 ng·mL−1 5.6×10−4 ng·mL−1 [160]

Sulfite SOD Fe3O4/GNPs Covalent Amperometric 5.0×10−7–1×10−3 M 1.5×10−7 M [161]

Xanthine XOD Fe3O4/CNTs Covalent Amperometric 2.5×10−7–3.5×10−6 M 6×10−8 M [162]

Urea Urease Fe3O4 Crosslinking Capacitive 1×10−5–3×10−4 M − [163]

Glucose GOx γ-Fe2O3 /PAH Crosslinking Conductometric − 3×10−6 M [164]

Ig G and interferon-γ

HRP γ-Fe2O3 Covalent Magnetoresistive 12 pg·mL−1 [165]

Glucose GOx Fe3O4 Covalent Potentiometric 1.0× 10−6–3.0× 10−2 M – [166]

Urea Urease PGMA/Fe3O4

Covalent Potentiometric 2.5×10−4–5.0×10−3 M 5×10−5 M [167]

SOD sulfite oxidase

Table 4 Optical enzyme biosensors based on iron oxide nanoparticles

Analyte Enzyme Matrix ImmobilizationMethod

Analytical method Performances Reference

Analytical range Limit ofdetection

Mycotoxinochratoxin A

Alkalinephosphatase

Fe2O3 Covalent Colorimetric − − [168]

Thrombin peroxidase mimic Fe3O4 − Colorimetric 1×10−9–1×10−7 M 1×10−9 M [169]

H2O2 peroxidase mimic Fe3O4 − Colorimetric 5×10−7–1.5×10−4 M 2.5×10−7 M [170]

Ethanol andmethanol

AOX Fe3O4 Entrapment Colorimetric 1×10−4–5×10−4 M 2.5×10−5 M [171]

Galactose GAO Fe3O4 Crosslinking Colorimetric 10–200 μg·mL−1 5 μg·mL−1 [172]

CA125 HRP Fe3O4 Covalent Electrochemiluminescence 0–10 mU·mL−1 8.0μU·mL−1 [173]

AFP α-AFP antibody Fe3O4 − Electrochemiluminescence 0.5 pg·mL−1–5.0 ng·mL−1 0.2 pg·mL−1 [174]

Glucose GOx γ-Fe2O3 Covalent Fluorescent 0–1.5×10−3 M 9×10−7 M [175]

Glucose GOx Fe3O4 Covalent Fluorimetric 1×10−3–2×10−2 M – [176]

Glucose GOx γ-Fe2O3/Rhodamine

Covalent Fluorimetric 1×10−8–1×10−6 M 1.5×10−9 M [177]

Biogenic amines DAO Fe3O4 Electrostatic Fluorimetric 2.5×10−5–3×10−5 M – [178]

H2O2 HRP Fe3O4 Crosslinking Fluorimetric 5.0×10−9–1×10−5 M 2.1×10−9 M [179]

Ochratoxin A Avidin Fe3O4 Covalent Luminescent 1×10−13–1×10−9 g·mL−1 1×10−13 g·mL−1 [180]

Thrombin Thrombin Fe3O4 Covalent Surface plasmon resonance 1× 10−11–1.5× 10−9 M 1.7× 10−11 M [181]

AFP α-AFP antibody Fe3O4 Covalent Surface plasmon resonance 1.0–200.0 ng·mL−1 0.65 ng·mL−1 [182]

AOX alcohol Oxidase; GAO galactose oxidase; DAO diamine oxidase

Enzymatic biosensors 11

constructed by modification with GOx as a biocomponent andnanostructured manganese oxide as a mediator [208–211].Yang reported the development of H2O2 biosensor by encap-sulation of HRP on methylene blue/layered manganese oxide[212] and polyquaternium-manganese oxide nanosheet nano-composite films [213]. Mohseni [214] studied thevoltammetric behavior of modified CPE with Cyt c andMn2O3 nanoparticles for H2O2 sensing. An amperometricbiosensor with amine oxidase and MnO2 for measuring bio-genic amines was developed and the enzymatically producedH2O2 was determined to quantify biogenic amines [215].

Nanostructured alumina is beneficial for high enzyme load-ing due to large surface area, porous morphology and hydro-philic property. Tyr [216], GOx [217, 218], AChE [219], HRP[220, 221] and galactosidase [222] were successfullyimmobilized on Al2O3. Very recently, Santos [223] developeda smart enzymatic sensor based on the PL of nanoporous anodicalumina (NAA) in the UV-visible range. The structure of NAAwas functionalized and activated for immobilizing trypsin in acontrolled manner (Fig. 10). Based on the change of PL spec-trum in each stage, it is possible to accurately detect andquantify the immobilized enzyme within the NAA structure.

Fig. 7 Schematic drawing (a) of the immunosensor fabrication process.(a) Dropping of chitosan-nanoAu hydrogel membrane; (b) immobilizingof anti-CEA; (c) the immunoreaction of CEA and anti-CEA; (d) incuba-tion of the solution containing multilabeled three-layer composite mag-netic conjugate resulting in an amplified signal-generating detection; (e)

separation of the immunocomplex using an external magnetic field per-mits the regeneration of the immunosensor. Calibration plots of thechanges of the current response via concentration of CEA (b) and AFP(c) with (a) and without (b) the addition of 1.25 mm glucose in pH 6.86PBS under optimal conditions. [ref. 137]

12 X. Shi et al.

Fig. 8 a Formation of the on-offmagnetic switching assay. Tyr-modified MNPs (size: 100 nm)are first mixed with multiwalledCNTs (MWCNTs). The resultingMNP–Tyr conjugate is washedwith phosphate-buffered saline(PBS) at pH 6.5 in the presence ofa magnetic field, and this givesrise to the MNP–Tyrbioconjugate, which is thenintroduced, along with MWCNT,onto the screen-printed electrode(SPE) to produce the SPE/MNP–Tyr–MWCNT switching system.b Current–time recordingsfor catechol obtained for theMNP–Tyr–MWCNT biosensor.[ref. 141]

Fig. 9 a Schematic illustration of the principle and process for ZrO2

adsorption-based EQCM immunoassays for Phospho-AChE, includingthe ME monolayer modification, ZrO2 film electro-deposition, Phospho-AChE capture, HRP-anti-AChE recognition, and HRP-catalytic precipi-tation of insoluble product towards amplified EQCM immunoassays. bCalibration curve plotted on a semi-log scale for detecting Phospho-

AChE in plasma samples, with a detection limit of 0.020 nM. The topinsert shows the comparison of final Δf responses of immunoassays forBSA (10 mg ml−1), AChE (0.1 nM) and Phospho-AChE (0.1 nM), andthe bottom insert manifests the real-time EQCMmonitoring for the directadsorptions of 0.5 nM a AChE and b Phospho-AChE onto the ZrO2-coated crystals [ref. 196]

Enzymatic biosensors 13

Besides the metal oxides mentioned above, CuO [224],Bi2O3 [225], CeO2 [226] nanoparticles were reported to beused for GOx immobilization and biosensor design. MgO[227], Ruthenium oxide [228] and Ni doped SnO2

nanoparticles [229] were also modified on electrode forHRP immobilization and H2O2 detection. Table 5 shows someother metal oxide nanoparticle-based enzyme biosensors.

Conclusions and perspectives

Over the past decades, tremendous efforts have beenperformed on MONPs based biosensing system. The continu-ing breakthroughs in the synthesis and modifications ofMONPs have brought new properties and new applicationsin biosensors with improved performance. MONPs not onlyretain the bioactivity of the immobilized enzyme but alsoenhance the sensing characteristics such as sensitivity, selec-tivity and low detection limit of the enzymatic biosensingsystem.

Although great advances in enzymatic biosensors based onMONPs have been achieved, there are many challenges in thedevelopment of biosensing system for real sample analysis.Up to now, almost all researches on MONPs based enzymaticbiosensors are still in laboratory. As for practical applications,few enzymatic biosensors appear to be commercially feasibleexcept for some blood glucose and hand-held immunosensors[255]. To our best knowledge, no commercial enzymatic

biosensor based on MONPs has been reported until now.Therefore, to commercialize the enzymatic biosensors basedon MONPs, efforts should be made to break some key tech-nical barriers such as controlling the morphology of MONPson device, realizing efficient enzyme immobilization andkeeping enzyme long-life bioactivity as well as reducingmatrix interference and sensor fouling. Firstly, controllingthe morphologies ofMONPs for biosensor design is necessarybecause the morphology of the nanosized material is one ofthe most important factors to determine the properties forbiosensor applications. MONPs usually demonstrate size-dependent as well as shape- and structure-dependent optical,electronic, thermal, and structural properties. Many syntheticmethods have been developed for controllable preparationMONPs, however, for practical biosensor design, the proce-dure of controllable synthesis and then deposition thenanoparticles on device is not a good choice because of thetendency aggregation of nanoparticles. Thus, in-situ synthesis,for example, electrochemical deposition, CVD, template syn-thesis and so on, might be powerful tools to grow controlledMONPs on device directly. Secondly, how to realize efficientenzyme immobilization is a very critical question that shouldbe addressed for the commercialization of biosensors.Conventional strategies, including physical or chemical im-mobilization, have their shortage for controlling the orienta-tion of the enzyme in order to implement efficient enzymeimmobilization and retain the long-life bioactivity of enzyme.Recently, the coupled use of site-directed mutagenesis and

Fig. 10 a Schematic diagramshowing the different stages of thefabrication process of the NAAenzymatic sensor. (a) As-produced NAA. (b) APTES-functionalized NAA. (c) GTA-activated NAA. (d) Trypsin-immobilized NAA. (e) Magnifiedview of the resulting NAAenzymatic sensor. b Change inthe effective optical thicknessincrement as a result of thedifferent fabrication stages.ΔOTeff at each fabrication stageof sample Bio (1). c Contourgraph of ΔOTeff as a function ofthe anodization time (tan) andpore widening time (tpw) forsamples Bio(1–9) [ref. 223]

14 X. Shi et al.

immobilization has been proved a very useful strategy thatcontrolling the orientation of the enzyme and improving en-zyme features from activity to stability [256]. It should befurther studied to test whether site-directed mutagenesisand immobilization enzymes in MONPs could improveenzyme features or not. Thirdly, for practical applicationof MONPs based enzymatic biosensors, problems asso-ciated with matrix interference and sensor foulingshould be resolved. Biosensors might have well perfor-mance under controlled environments or laboratory sam-ples, however, in real sample analysis, the matrix inter-ference and sensor fouling are the detrimental factors

for commercialization. Especially, the large surface toarea for nanoparticles might lead to more serious sensorfouling. Thus, more effort should be made to solvethese problems.

Despite these numerous challenges, MONPs based enzy-matic biosensing system is considered to be one of the mostpromising tools for food safety detection, environmental pol-lution control, point-of-care testing and so on. With the ad-vancement of nanotechnology and biotechnology, more enzy-matic biosensing platforms based on MONPs will beestablished and new breakthroughs in practical applicationswill be realized.

Table 5 Other MONPs for enzyme immobilization and the performance of enzymatic biosensors

Analyte Enzyme MONPs matrix ImmobilizationMethod

Analyticalmethod

Performances References

Analytical range Limit ofdetection

H2O2 HRP ZrO2/Silica Entrapment Amperometric 7.8×10−7–3.2×10−3 M 3.2×10−7 M [230]

H2O2 Hb ZrO2/Collagen Entrapment Amperometric 8×10−7–1.32×10−4 M 1.2×10−7 M [231]

H2O2 Catalase NiO − Amperometric 1×10−6–1×10−3 M 0.96(± 0.05)mM

[232]

H2O2 HRP RuO2 Crosslinking Amperometric 5.09×10−3–1.5×10−2 M – [228]

H2O2 HRP SnO2/Ni Physical adsorption Amperometric 1.0×10−7–3.0×10−4 M 4.3×10−8 M [229]

H2O2 Mb ZrO2/CNTs Electrochemicaldeposition

Amperometric 2×10−6–1×10−3 M 6×10−7 M [233]

H2O2 HRP ZrO2/Collagen Entrapment Amperometric 1×10−6–7.3×10−5 M 2.5×10−7 M [234]

H2O2 ChOx MnO2 − Amperometric 1.0×10−5–2.1×10−3 M − [235]

H2O2 Catalase Al2O3/MB Entrapment Amperometric 1×10−5–1×10−4 M − [236]

H2O2 HRP Al2O3 Entrapment Amperometric 1.76×10−3–5.5×10−2 M 1.1×10−4 M [237]

H2O2 HRP tt-MgO Physical adsorption Amperometric 1×10−6–4.5×10−4 M 3×10−7 M [227]

H2O2 HRP Bi2O3/CNTs Electrostatic Amperometric 8.34×10−3–2.888×10−2 M − [238]

Glucose GOx NiO − Amperometric 3×10−5–5×10−3 M. 2.4×10−5 M [239]

Glucose GOx MnO2 Physical adsorption FET 2.5×10−5–1.9×10−3 M 3.5×10−9 M [207]

Glucose GOx MnO2 Entrapment Amperometric 9×10−7–2.73×10−3 M 1.8×10−7 M [240]

Glucose GOx Al2O3 Entrapment Amperometric 4×10−8–2.19×10−3 M 1×10−5 M [241]

Glucose GOx Al2O3 Crosslinking Amperometric 2.5×10−4–8×10−3 M − [242]

Glucose GOx CeO2 Physical adsorption Amperometric 2×10−3–2.6×10−2 M 1×10−4 M [243]

Lactate LOD MnO2 Electrostatic FET 1×10−5–3.6×10−3 M 8×10−6 M [244]

Lactate LOD MnO2 Physical adsorption Amperometric 2×10−4–5×10−3 M – [245]

Bilirubin BOD ZrO2/Silica Covalent Amperometric 2×10−8–2.5×10−4 M 1×10−10 M [246]

Cholesterol ChOx NiFe2O4/CuO/FeO

Electrostatic Amperometric 50–5,000 mg·L−1 313 mg·L−1 [247]

Cholesterol ChOx NiO Electrostatic Amperometric 10–400 mg·dL−1 43.4 mg·dL−1 [201]

Cyt c Cyt coxidase

NiO/CNTs Physical adsorption Amperometric 5×10−12–5×10−7 M 5×10−12 M [248]

Ethanol ADH NiO Physical adsorption Amperometric 1.6×10−3–3.8×10−2 M – [249]

Phenoliccompounds

Laccase MnO2/CNTs/PANI

Covalent Amperometric 1×10−7–1×10−5 M and 1× 10−5–5×10−4 M

4×10−8 M [250]

Phenols Tyr Al2O3 Entrapment Amperometric 5×10−7–3×10−5 M 3×10−7 M. [251]

SRB HRP MnO2 Covalent Amperometric 1.8×105–1.8×108 CFU·mL−1 − [252]

Urea Urease NiO Physical adsorption Amperometric 8.3×10−4–1.665×10−2 M – [204]

Urea Urease Al2O3 Entrapment Amperometric 5×10−7–3×10−3 M 2×10−7 M [253]

Uric acid Uricase CuO/Pt Physical adsorption Amperometric 5×10−5–1×10−3 M − [254]

ADH alcohol dehydrogenase

Enzymatic biosensors 15

Acknowledgments This work was supported by the National NaturalScience Foundation of China (No. 21175046), New Century ExcellentTalents in University (No. NCET-09-0357) and Open Foundation ofShanghai Key Laboratory of Green Chemistry and Chemical Process.

References

1. Debecker DP, Mutin PH (2012) Non-hydrolytic sol-gel routes toheterogeneous catalysts. Chem Soc Rev 41(9):3624–3650

2. Meyer J, Hamwi S, Kroeger M, Kowalsky W, Riedl T, Kahn A(2012) Transition metal oxides for organic electronics: energetics,device physics and applications. Adv Mater 24(40):5408–5427

3. Jiang J, Li Y, Liu J, Huang X, Yuan C, Lou XW (2012) Recentadvances in metal oxide-based electrode architecture design forelectrochemical energy storage. Adv Mater 24(38):5166–5180

4. Shaidarova LG, Budnikov GK (2008) Chemically modified elec-trodes based on noble metals, polymer films, or their composites inorganic voltammetry. J Anal Chem 63(10):922–942

5. Shaidarova LG, Gedmina AV, Chelnokova IA, Budnikov GK(2011) Selective determination of paracetamol and acetylsalicylicacid on electrode modified with a mixed-valent film of rutheniumoxide-ruthenium cyanide. Russ J Appl Chem 84(4):620–627

6. Haun JB, Yoon T-J, Lee H, Weissleder R (2010) Magnetic nano-particle biosensors. Wiley Interdiscip Rev 2(3):291–304

7. Xu Y, Wang E (2012) Electrochemical biosensors based on mag-netic micro/nano particles. Electrochim Acta 84:62–73

8. Arya SK, Saha S, Ramirez-Vick JE, Gupta V, Bhansali S, Singh SP(2012) Recent advances in ZnO nanostructures and thin films forbiosensor applications: review. Anal Chim Acta 737:1–21

9. Yakimova R, Selegard L, Khranovskyy V, Pearce R, Spetz AL,Uvdal K (2012) ZnO materials and surface tailoring for biosensing.Front Biosci (Elite Ed) 4:254–278

10. Solanki PR, Kaushik A, Agrawal VV, Malhotra BD (2011) Nano-structured metal oxide-based biosensors. NPG Asia Mater 3(1):17–24

11. Beek WJE, Wienk MM, RaJ J (2004) Efficient hybrid solar cellsfrom zinc oxide nanoparticles and a conjugated polymer. AdvMater16(12):1009–1013

12. Umar A, Rahman MM, Kim SH, Hahn YB (2008) Zinc oxidenanonail based chemical sensor for hydrazine detection. ChemCommun 44(2):166–168

13. Zeng H, Cai W, Liu P, Xu X, Zhou H, Klingshirn C, Kalt H (2008)ZnO-based hollow nanoparticles by selective etching: eliminationand reconstruction of metal-semiconductor interface, improvementof blue emission and photocatalysis. ACS Nano 2(8):1661–1670

14. Dolcet P, Casarin M, Maccato C, Bovo L, Ischia G, Gialanella S,Mancin F, Tondello E, Gross S (2012) Miniemulsions as chemicalnanoreactors for the room temperature synthesis of inorganic crys-talline nanostructures: ZnO colloids. J Mater Chem 22(4):1620–1626

15. Monge M, Kahn ML, Maisonnat A, Chaudret B (2003) Room-temperature organometallic synthesis of soluble and crystallineZnO nanoparticles of controlled size and shape. Angew Chem IntEd 42(43):5321–5324

16. Zhao Z, Lei W, Zhang X, Wang B, Jiang H (2010) ZnO-basedamperometric enzyme biosensors. Sensors 10(2):1216–1231

17. Dai ZH, Shao GJ, Hong JM, Bao JC, Shen J (2009) Immobilizationand direct electrochemistry of glucose oxidase on a tetragonalpyramid-shaped porous ZnO nanostructure for a glucose biosensor.Biosens Bioelectron 24(5):1286–1291

18. Palanisamy S, Vilian ATE, Chen SM (2012) Direct electrochemistryof glucose oxidase at reduced graphene oxide/zinc oxide composite

modified electrode for glucose sensor. Int J Electron Sc 7(3):2153–2163

19. Hu FX, Chen SH, Wang CY, Yuan R, Chai YQ, Xiang Y, Wang C(2011) ZnO nanoparticle and multiwalled carbon nanotubes forglucose oxidase direct electron transfer and electrocatalytic activityinvestigation. J Mol Catal B 72(3–4):298–304

20. Zhao ZW, Chen XJ, Tay BK, Chen JS, Han ZJ, Khor KA (2007) Anovel amperometric biosensor based on ZnO : Co nanoclusters forbiosensing glucose. Biosens Bioelectron 23(1):135–139

21. Yang C, Xu CX, Wang XM (2012) ZnO/Cu nanocomposite: aplatform for direct electrochemistry of enzymes and biosensingapplications. Langmuir 28(9):4580–4585

22. Wang YT, Yu L, Zhu ZQ, Zhang J, Zhu JZ, Fan CH (2009)Improved enzyme immobilization for enhanced bioelectrocatalyticactivity of glucose sensor. Sensors Actuators B 136(2):332–337

23. Bin F, Cuihong Z, Guangfeng W, Meifang W, Yulan J (2011) Aglucose oxidase immobilization platform for glucose biosensorusing ZnO hollow nanospheres. Sensors Actuators B 155(1):304–310310

24. Umar A, RahmanMM,VaseemM, HahnYB (2009) Ultra-sensitivecholesterol biosensor based on low-temperature grown ZnOnanoparticles. Electrochem Commun 11(1):118–121

25. Wang CY, Tan XR, Chen SH, Yuan R, Hu FX, Yuan DH, Xiang Y(2012) Highly-sensitive cholesterol biosensor based on platinum-gold hybrid functionalized ZnO nanorods. Talanta 94:263–270

26. Batra N, Tomar M, Gupta V (2012) Realization of an efficientcholesterol biosensor using ZnO nanostructured thin film. Analyst137(24):5854–5859

27. Singh SP, Arya SK, Pandey P, Malhotra BD, Saha S, Sreenivas K,Gupta V (2007) Cholesterol biosensor based on rf sputtered zincoxide nanoporous thin film. Appl Phys Lett 91(6):063901

28. Negahdary M, Asadi A, Mehrtashfar S, Imandar M, Akbari-Dastjerdi H, Salahi F, Jamaleddini A, Ajdary M (2012) Abiosensor for determination of H2O2 by use of HRP enzymeand modified CPE with ZnO NPs. Int J Electron Sc 7(6):5185–5194

29. Xiang C, Zou Y, Sun LX, Xu F (2009) Direct electrochemistry andenhanced electrocatalysis of horseradish peroxidase based on flow-erlike ZnO-gold nanoparticle-Nafion nanocomposite. Sensors Ac-tuators B 136(1):158–162

30. Zhang YW, Zhang Y, Wang H, Yan B, Shen GL, Yu RQ (2009) Anenzyme immobilization platform for biosensor designs of directelectrochemistry using flower-like ZnO crystals and nano-sizedgold particles. J Electroanal Chem 627(1–2):9–14

31. Li YF, Liu ZM, Liu YL, Yang YH, Shen GL, Yu RQ (2006) Amediator-free phenol biosensor based on immobilizing tyrosinase toZnO nanoparticles. Anal Biochem 349(1):33–40

32. Liu ZM, Liu YL, Yang HF, Yang Y, Shen GL, Yu RQ (2005) Amediator-free tyrosinase biosensor based on ZnO sol-gel matrix.Electroanalysis 17(12):1065–1070

33. Ansari SG, Wahab R, Ansari ZA, Kim YS, Khang G, Al HA, ShinHS (2009) Effect of nanostructure on the urea sensing properties ofsol-gel synthesized ZnO. Sensors Actuators B 137(2):566–573

34. Ali A, Ansari AA, Kaushik A, Solanki PR, Barik A, Pandey MK,Malhotra BD (2009) Nanostructured zinc oxide film for urea sensor.Mater Lett 63(28):2473–2475

35. Zhang FF,WangXL, Ai SY, Sun ZD,WanQ, Zhu ZQ, XianYZ, JinLT, Yamamoto K (2004) Immobilization of uricase on ZnOnanorods for a reagentless uric acid biosensor. Anal Chim Acta519(2):155–160

36. Wang YT, Yu L, Zhu ZQ, Zhang J, Zhu JZ (2009) Novel uric acidsensor based on enzyme electrode modified by ZnO nanoparticlesand multiwall carbon nanotubes. Anal Lett 42(5):775–789

37. Zhao YG, Yan XQ, Kang Z, Lin P, Fang XF, Lei Y, Ma SW, ZhangY (2013) Highly sensitive uric acid biosensor based on individualzincoxide micro/nanowires. Microchim Acta 180:759–766

16 X. Shi et al.

38. Devi R, Yadav S, Pundir CS (2012) Amperometric determination ofxanthine in fish meat by zinc oxide nanoparticle/chitosan/multiwalled carbon nanotube/polyaniline composite film boundxanthine oxidase. Analyst 137(3):754–759

39. Devi R, Thakur M, Pundir CS (2011) Construction and applicationof an amperometric xanthine biosensor based on zinc oxidenanoparticles-polypyrrole composite film. Biosens Bioelectron26(8):3420–3426

40. Yang MH, Yang YH, Yang Y, Shen GL, Yu RQ (2005)Microbiosensor for acetylcholine and choline based onelectropolymerization/sol-gel derived composite membrane. AnalChim Acta 530(2):205–211

41. Wang YT, Yu L, Wang J, Lou L, Du WJ, Zhu ZQ, Peng H, Zhu JZ(2011) A novel l-lactate sensor based on enzyme electrode modifiedwith ZnO nanoparticles and multiwall carbon nanotubes. JElectroanal Chem 661(1):8–12

42. Guan H, Chi D, Yu J (2011) Photoelectrochemical acetylcholines-terase biosensor incorporating zinc oxide nanoparticles. Adv MaterRes 183–185:1701–1706

43. Yadav S, Devi R, Kumar A, Pundir CS (2011) Tr-enzyme function-alized ZnO-NPs/CHIT/c-MWCNT/PANI composite film for am-perometric determination of creatinine. Biosens Bioelectron 28(1):64–70

44. Jia XD, Lu F, Liu Y, Zhu JJ (2011) Synthesis of flower-like zincoxide nanocrystals and their electrochemical biosensing. Chin JInorg Chem 27(6):1150–1154

45. Zhu X, Yuri I, Gan X, Suzuki I, Li G (2007) Electrochemical studyof the effect of nano-zinc oxide on microperoxidase and its appli-cation to more sensitive hydrogen peroxide biosensor preparation.Biosens Bioelectron 22(8):1600–1604

46. Lei Y, Yan X, Zhao J, Liu X, Song Y, Luo N, Zhang Y (2011)Improved glucose electrochemical biosensor by appropriate immo-bilization of nano-ZnO. Collods Surf B 82(1):168–172

47. Ahmad M, Pan C, Gan L, Nawaz Z, Zhu J (2010) Highly sensitiveamperometric cholesterol biosensor based on Pt-incorporatedfullerene-like ZnO nanospheres. J Phys Chem C 114(1):243–250

48. Shukla SK, Deshpande SR, Shukla SK, Tiwari A (2012) Fabricationof a tunable glucose biosensor based on zinc oxide/chitosan-graft-poly(vinyl alcohol) core-shell nanocomposite. Talanta 99:283–287

49. Ren XL, Chen D, Meng XW, Tang FQ, Hou XQ, Han D, Zhang L(2009 ) Z inc ox ide nanopa r t i c l e s / g l u co s e ox i da s ephotoelectrochemical system for the fabrication of biosensor. JColloid Interface Sci 334(2):183–187

50. Kim KE, Kim TG, Sung YM (2012) Enzyme-conjugated znonanocrystals for collisional quenching-based glucose sensing.Crystengcomm 14(8):2859–2865

51. Cosnier S, Gondran C, Senillou A, Gratzel M, Vlachopoulos N(1997)Mesoporous TiO2 films: new catalytic electrodematerials forfabricating amperometric biosensors based on oxidases. Electro-analysis 9(18):1387–1392

52. Yu JH, Liu SQ, Ju HX (2003) Glucose sensor for flow injectionanalysis of serum glucose based on immobilization of glucoseoxidase in titania sol-gel membrane. Biosens Bioelectron 19(4):401–409

53. Maniruzzaman M, Jang SD, Kim J (2012) Titanium dioxide-cellulose hybrid nanocomposite and its glucose biosensor applica-tion. Mater Sci Eng B 177(11):844–848

54. Cao H, Zhu Y, Tang L, Yang X, Li C (2008) A glucose biosensorbased on immobilization of glucose oxidase into 3D macroporousTiO2. Electroanalysis 20(20):2223–2228

55. Lee SY, Matsuno R, Ishihara K, Takai M (2013) Direct electrontransfer with enzymes on nanofiliform titanium oxide films withelectron-transport ability. Biosens Bioelectron 41:289–293

56. Wei N, Xin X, Du J, Li J (2011) A novel hydrogen peroxidebiosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-

doped titanium dioxide (GTD) film. Biosens Bioelectron 26(8):3602–3607

57. Chavhan PM, Reddy V, Kim C (2012) Nanostructured titaniumoxide platform for application to ascorbic acid detection. Int JElectron Sc 7(6):5420–5428

58. Majidi MR, Asadpour ZK, Gholizadeh S (2010) Nanobiocompositemodified carbon-ceramic electrode based on nano-TiO2-plant tissueand its application for electrocatalytic oxidation of dopamine. Elec-troanalysis 22(15):1772–1780

59. Tasviri M, Rafiee PHA, Ghourchian H, Gholami MR (2011) Aminefunctionalized TiO2 coated on carbon nanotube as a nanomaterialfor direct electrochemistry of glucose oxidase and glucosebiosensing. J Mol Catal B 68(2):206–210

60. Jang HD, Kim SK, Chang H, Roh KM, Choi JW, Huang JX (2012)A glucose biosensor based on TiO2-graphene composite. BiosensBioelectron 38(1):184–188

61. Zhang MH, Yuan R, Chai YQ, Li WJ, Zhong H, Wang C (2011)Glucose biosensor based on titanium dioxide-multiwall carbonnanotubes-chitosan composite and functionalized goldnanoparticles. Bioprocess Biosyst Eng 34(9):1143–1150

62. Li QW, Luo GA, Feng J, Zhou Q, Zhang L, Zhu YF (2001)Amperometric detection of glucose with glucose oxidase absorbedon porous nanocrystalline TiO2 film. Electroanalysis 13(5):413–416

63. Sousa CP, Polo AS, Torresi RM, De Torresi SIC, Alves WA (2010)Chemical modification of a nanocrystalline TiO2 film for efficientelectric connection of glucose oxidase. J Colloid Interface Sci346(2):442–447

64. Li Y, Liu XY, Yuan HY, Xiao D (2009) Glucose biosensor based onthe room-temperature phosphorescence of TiO2/SiO2 nanocompos-ite. Biosens Bioelectron 24(12):3706–3710

65. Chou JC, Yang HY, Chen CW (2010) Glucose biosensor ofruthenium-doped TiO2 sensing electrode by co-sputtering system.Microelectron Reliab 50(5):753–756

66. Zhang XJ, Wang GF, Huang Y, Yu LT, Fang B (2011) Copper(II)doped nanoporous TiO2 composite based glucose biosensor. AnalMethods 3(11):2611–2615

67. Ding SN, Gao BH, Shan D, Sun YM, Cosnier S (2013) TiO2

nanocrystals electrochemiluminescence quenching by biologicalenlarged nanogold particles and its application for biosensing.Biosens Bioelectron 39(1):342–345

68. Chen X, Dong SJ (2003) Sol-gel-derived titanium oxide/copolymercomposite based glucose biosensor. Biosens Bioelectron 18(8):999–1004

69. Cosnier S, Senillou A, Gratzel M, Comte P, Vlachopoulos N,Renault NJ, Martelet C (1999) A glucose biosensor based onenzyme entrapment within polypyrrole films electrodeposited onmesoporous titanium dioxide. J Electroanal Chem 469(2):176–181

70. Doong RA, Shih HM (2010) Array-based titanium dioxide biosen-sors for ratiometric determination of glucose, glutamate and urea.Biosens Bioelectron 25(6):1439–1446

71. Zhang Y, He PL, Hu NF (2004) Horseradish peroxidaseimmobilized in TiO2 nanoparticle films on pyrolytic graphite elec-trodes: direct electrochemistry and bioelectrocatalysis. ElectrochimActa 49(12):1981–1988

72. Zhong HA, Yuan R, Chai YQ, Li WJ, Zhang Y, Wang CY (2011)Amperometric biosensor for hydrogen peroxide based on horserad-ish peroxidase onto gold nanowires and TiO2 nanoparticles.Bioprocess Biosyst Eng 34(8):923–930

73. Wang Y, Ma XL, Wen Y, Xing YY, Zhang ZR, Yang HF (2010)Direct electrochemistry and bioelectrocatalysis of horseradish per-oxidase based on gold nano-seeds dotted TiO2 nanocomposite.Biosens Bioelectron 25(11):2442–2446

74. Li Q, Cheng K, WengWJ, Du PY, Han GR (2012) Highly sensitivehydrogen peroxide biosensors based on TiO2 nanodots/ITO elec-trodes. J Mater Chem 22(18):9019–9026

Enzymatic biosensors 17

75. Tan SW, Tan XC, Xu J, Zhao DD, Zhang JL, Liu L (2011) A novelhydrogen peroxide biosensor based on sol-gel poly (vinyl alcohol)(PVA)/(titanium dioxide)TiO2 hybrid material. Anal Methods 3(1):110–115

76. Xu X, Zhao JQ, Jiang DC, Kong JL, Liu BH, Deng JQ (2002) TiO2

sol-gel derived amperometric biosensor for H2O2 on theelectropolymerized phenazine methosulfate modified electrode.Anal Bioanal Chem 374(7–8):1261–1266

77. Lu HY, Yang J, Rusling JF, Hu NF (2006) Vapor-surface sol-geldeposition of titania alternated with protein adsorption for assemblyof electroactive, enzyme-active films. Electroanalysis 18(4):379–390

78. Li WJ, Yuan R, Chai YQ, Hong CL, Zhuo Y (2008) Reagentlesselectrochemical hydrogen peroxide biosensor based on toluidineblue-derived organic material and functionalized gold nanoparticles.J Electrochem Soc 155(5):F97–F103

79. Zhou H, Liu L, Yin K, Liu SL, Li GX (2006) Electrochemicalinvestigation on the catalytic ability of tyrosinase with the effectof nano titanium dioxide. Electrochem Commun 8(7):1168–1172

80. Chen X, Cheng GJ, Dong SJ (2001) Amperometric tyrosinasebiosensor based on a sol-gel-derived titanium oxide-copolymercomposite matrix for detection of phenolic compounds. Analyst126(10):1728–1732

81. Lee YJ, Lyn YK, Choi HN, Lee WY (2007) Amperometric tyros-inase biosensor based on carbon nanotube-titania-Nafion compositefilm. Electroanalysis 19(10):1048–1054

82. Zhang T, Tian BZ, Kong JL, Yang PY, Liu BH (2003) A sensitivemediator-free tyrosinase biosensor based on an inorganic-organichybrid titania sol-gel matrix. Anal Chim Acta 489(2):199–206

83. Doong RA, Shih HM (2006) Glutamate optical biosensor based onthe immobilization of glutamate dehydrogenase in titanium dioxidesol-gel matrix. Biosens Bioelectron 22(2):185–191

84. Yang HC, Yuan R, Chai YQ, Su HL, Zhuo Y, Jiang W, Song ZJ(2011) Electrochemical immunosensor for human chorionic gonad-otropin based on horseradish peroxidase-functionalized prussianblue-carbon nanotubes/gold nanocomposites as labels for signalamplification. Electrochim Acta 56(5):1973–1980

85. Cheng JJ, Di JW, Hong JH, Yao KA, Sun YB, Zhuang JY, Xu Q,Zheng HE, Bi SP (2008) The promotion effect of titaniananoparticles on the direct electrochemistry of lactate dehydroge-nase sol-gel modified gold electrode. Talanta 76(5):1065–1069

86. Kafi AKM, Chen AC (2009) A novel amperometric biosensor forthe detection of nitrophenol. Talanta 79(1):97–102

87. Wang K, Li HN, Wu J, Ju C, Yan JJ, Liu Q, Qiu BJ (2011) TiO2-decorated graphene nanohybrids for fabricating an amperometricacetylcholinesterase biosensor. Analyst 136(16):3349–3354

88. Jia JB, Zhang SP, Wang P, Wang HJ (2012) Degradation of highconcentration 2,4-dichlorophenol by simultaneous photocatalytic-enzymatic process using TiO2/UVand laccase. J Hazard Mater 205:150–155

89. Su HL, Yuan R, Chai YQ, Zhuo Y (2012) Enzyme-nanoparticleconjugates at oil-water interface for amplification of electrochemicalimmunosensing. Biosens Bioelectron 33(1):288–292

90. Zhuo Y, Chai YQ, Yuan R, Mao L, Yuan YL, Han J (2011) Glucoseoxidase and ferrocene labels immobilized at Au/TiO2 nanocompos-ites with high load amount and activity for sensitiveimmunoelectrochemical measurement of progrp biomarker. BiosensBioelectron 26(9):3838–3844

91. Emregul E, KocabayO, Derkus B, Yumak T, Emregul KC, Sinag A,Polat K (2013) A novel carboxymethylcellulose-gelatin-titaniumdioxide-superoxide dismutase biosensor; electrochemical propertiesof carboxymethylcellulose-gelatin-titanium dioxide-superoxidedismutase. Bioelectrochemistry 90:8–17

92. Wei YY, Li Y, Qu YH, Xiao F, Shi GY, Jin LT (2009) A novelbiosensor based on photoelectro-synergistic catalysis for flow-injection analysis system/amperometric detection of organophos-phorous pesticides. Anal Chim Acta 643(1–2):13–18

93. Xie Q, Zhao Y, Chen X, Liu H, Evans DG, Yang W (2011)Nanosheet-based titania microspheres with hollow core-shell struc-ture encapsulating horseradish peroxidase for a mediator-free bio-sensor. Biomaterials 32(27):6588–6594

94. Li YJ, Ma MJ, Zhu JJ (2012) Dual-signal amplification strategy forultrasensitive photoelectrochemical immunosensing of alpha-fetoprotein. Anal Chem 84(23):10492–10499

95. Xu P, Zeng GM, Huang DL, Feng CL, Hu S, Zhao MH, Lai C, WeiZ, Huang C, Xie GX, Liu ZF (2012) Use of iron oxidenanomaterials in wastewater treatment: a review. Sci Total Environ424:1–10

96. Horak D, Babic M, Mackova H, Benes MJ (2007) Preparation andproperties of magnetic nano- and microsized particles for biologicaland environmental separations. J Sep Sci 30(11):1751–1772

97. Lee N, Hyeon T (2012) Designed synthesis of uniformly sized ironoxide nanoparticles for efficient magnetic resonance imaging con-trast agents. Chem Soc Rev 41(7):2575–2589

98. Liu F, Laurent S, Fattahi H, Elst LV, Muller RN (2011)Superparamagnetic nanosystems based on iron oxide nanoparticlesfor biomedical imaging. Nanomedicine 6(3):519–528

99. Arruebo M, Fernandez-Pacheco R, Ibarra MR, Santamaria J (2007)Magnetic nanoparticles for drug delivery. Nano Today 2(3):22–32

100. Kim JE, Shin JY, Cho MH (2012) Magnetic nanoparticles: anupdate of application for drug delivery and possible toxic effects.Arch Toxicol 86(5):685–700

101. Veiseh O, Gunn JW, Zhang M (2010) Design and fabrication ofmagnetic nanoparticles for targeted drug delivery and imaging. AdvDrug Deliv Rev 62(3):284–304

102. Tassa C, Shaw SY, Weissleder R (2011) Dextran-coated iron oxidenanoparticles: a versatile platform for targeted molecular imaging,molecular diagnostics, and therapy. Acc Chem Res 44(10):842–852

103. Kievit FM, Zhang M (2011) Surface engineering of iron oxidenanoparticies for targeted cancer therapy. Acc Chem Res 44(10):853–862

104. Netto CGCM, Toma HE, Andrade LH (2013) Superparamagneticnanoparticles as versatile carriers and supporting materials for en-zymes. J Mol Catal B 85–86:71–92

105. Sandhu A, Handa H, Abe M (2010) Synthesis and applications ofmagnetic nanoparticles for biorecognition and point of care medicaldiagnostics. Nanotechnology 21(44):442001

106. Wang SX, Li G (2008) Advances in giant magnetoresistance bio-sensors with magnetic nanoparticle tags: review and outlook. IEEETrans Magn 44(7):1687–1702

107. Jana NR, Chen YF, Peng XG (2004) Size- and shape-controlledmagnetic (Cr, Mn, Fe, Co, Ni) oxide nanocrystals via a simple andgeneral approach. Chem Mater 16(20):3931–3935

108. Li JJ, Yuan R, Chai YQ, Che X (2010) Fabrication of a novelglucose biosensor based on Pt nanoparticles-decorated iron oxide-multiwall carbon nanotubes magnetic composite. J Mol Catal B66(1–2):8–14

109. Krishna R, Titus E, Chandra S, BardhanNK, Krishna R, Bahadur D,Gracio J (2012) Fabrication of a glucose biosensor based on citricacid assisted cobalt ferrite magnetic nanoparticles. J NanosciNanotechnol 12(8):6631–6638

110. Ozdemir C, Akca O, Medine EI, Demirkol DO, Unak P, Timur S(2012) Biosensing applications of modified core-shell magneticnanoparticles. Food Anal Methods 5(4):731–736

111. Baby TT, Ramaprabhu S (2010) SiO2 coated Fe3O4 magnetic nano-particle dispersed multiwalled carbon nanotubes based amperomet-ric glucose biosensor. Talanta 80(5):2016–2022

112. Li JP, Chen XZ (2008) A novel glucose sensor based on sensitivefilm composed of Fe3O4/Au/GOXmagnetic particulates. Acta ChimSin 66(1):84–90

113. Yu JJ, Tu JX, Zhao FQ, ZengBZ (2010) Direct electrochemistry andbiocatalysis of glucose oxidase immobilized on magneticmesoporous carbon. J Solid State Electrochem 14(9):1595–1600

18 X. Shi et al.

114. Zou C, Fu YC, Xie QJ, Yao SZ (2010) High-performance glucoseamperometric biosensor based on magnetic polymericbionanocomposites. Biosens Bioelectron 25(6):1277–1282

115. Jimenez J, Sheparovych R, Pita M, Garcia AN, Dominguez E,Minko S, Katz E (2008) Magneto-induced self-assembling of con-ductive nanowires for biosensor applications. J Phys Chem C112(19):7337–7344

116. Wang AJ, Li YF, Li ZH, Feng JJ, Sun YL, Chen JR (2012)Amperometric glucose sensor based on enhanced catalytic reduc-tion of oxygen using glucose oxidase adsorbed onto core-shellFe3O4@silica@Au magnetic nanoparticles. Mater Sci Eng CMaterBiol Appl 32(6):1640–1647

117. Peng HP, Liang RP, Zhang L, Qiu JD (2013) Facile preparation ofnovel core-shell enzyme-au-polydopamine-Fe3O4 magneticbionanoparticles for glucosesensor. Biosens Bioelectron 42:293–299

118. Chen X, Zhu J, Chen Z, Xu C, Wang Y, Yao C (2011) A novelbienzyme glucose biosensor based on three-layer Au-Fe3O4@SiO2

magnetic nanocomposite. Sensors Actuators B 159(1):220–228119. Li JP, Wei XP, Yuan YH (2009) Synthesis of magnetic nanoparticles

composed by prussian blue and glucose oxidase for preparinghighly sensitive and selective glucose biosensor. Sensors ActuatorsB 139(2):400–406406

120. Jianding Q, Huaping P, Ruping L (2007) Ferrocene-modifiedFe3O4@SiO2 magnetic nanoparticles as building blocks for con-struction of reagentless enzyme-based biosensors. ElectrochemCommun 9(11):2734–27382738

121. Yang LQ, Ren XL, Tang FQ, Zhang L (2009) A practical glucosebiosensor based on Fe3O4 nanoparticles and chitosan/Nafion com-posite film. Biosens Bioelectron 25(4):889–895

122. Kim MI, Ye Y, Won BY, Shin S, Lee J, Park HG (2011) A highlyefficient electrochemical biosensing platform by employing con-ductive nanocomposite entrapping magnetic nanoparticles and ox-idase in mesoporous carbon foam. Adv Funct Mater 21(15):2868–2875

123. Tan XC, Zhang JL, Tan SW, Zhao DD, Huang ZW, Mi Y, HuangZY (2009) Amperometric hydrogen peroxide biosensor based onhorseradish peroxidase immobilized on Fe3O4/chitosan modifiedglassy carbon electrode. Electroanalysis 21(13):1514–1520

124. Yalciner F, Cevik E, Senel M, Baykal A (2011) Development of anamperometric hydrogen peroxide biosensor based on the immobili-zation of horseradish peroxidase onto nickel ferrite nanoparticle-chitosan composite. Nano-Micro Lett 3(2):91–98

125. Zhang HL, Lai GS, Han DY, Yu AM (2008) An amperometrichydrogen peroxide biosensor based on immobilization of horserad-ish peroxidase on an electrode modified with magnetic dextranmicrospheres. Anal Bioanal Chem 390(3):971–977

126. Qu S, Huang F, Chen G, Yu S, Kong J (2007) Magnetic assembledelectrochemical platform using Fe2O3 filled carbon nanotubes andenzyme. Electrochem Commun 9(12):2812–2816

127. Peng HP, Liang RP, Qiu JD (2011) Facile synthesis ofFe3O4@Al2O3 core-shell nanoparticles and their application tothe highly specific capture of heme proteins for direct electrochem-istry. Biosens Bioelectron 26(6):3005–3011

128. Qiu JD, Peng HP, Liang RP, Xia XH (2010) Facile preparation ofmagnetic core-shell Fe3O4@Au nanoparticle/myoglobin biofilm fordirect electrochemistry. Biosens Bioelectron 25(6):1447–1453

129. Lai GS, Zhang HL, Han DY (2008) A novel hydrogen peroxidebiosensor based on hemoglobin immobilized on magnetic chitosanmicrospheres modified electrode. Sensors Actuators B Chem129(2):497–503503

130. Cui RJ, Yin F, Zhou LJ, Pan HC (2011) Direct electrochemistry ofhemoglobin based on Fe3O4@SiO2 nanoparticles modified elec-trode. Chin J Chem 29(11):2481–2486

131. Won YH, Aboagye D, Jang HS, Jitianu A, Stanciu LA (2010) Core/shell nanoparticles as hybrid platforms for the fabrication of ahydrogen peroxide biosensor. J Mater Chem 20(24):5030–5034

132. Yang HW, Hua MY, Chen SL, Tsai RY (2013) Reusable sensorbased on high magnetization carboxyl-modified graphene oxidewith intrinsic hydrogen peroxide catalytic activity for hydrogenperoxide and glucose detection. Biosens Bioelectron 41:172–179

133. Zhang ZX, Zhu H, Wang XL, Yang XR (2011) Sensitive electro-chemical sensor for hydrogen peroxide using Fe3O4 magneticnanoparticles as a mimic for peroxidase. Microchim Acta 174(1–2):183–189

134. Zhang LH, Zhai YM, Gao N, Wen D, Dong SJ (2008) SensingH2O2 with layer-by-layer assembled Fe3O4-PDDA nanocompositefilm. Electrochem Commun 10(10):1524–1526

135. Jin HJ, Gan N, Hou JG, Hu FT, Cao YT, Zheng L, Guo ZY (2012)Signal amplification of electrochemical elisa for the detection ofalpha-fetoprotein using core-shell Fe3O4@Au nanoparticles as la-bels. Sens Lett 10(3–4):886–893

136. Tang DP, Yuan R, Chai YQ (2006) Direct electrochemical immu-noassay based on immobilization of protein-magnetic nanoparticlecomposites on to magnetic electrode surfaces by sterically enhancedmagnetic field force. Biotechnol Lett 28(8):559–565

137. Zhuo Y, Yuan PX, Yuan R, Chai YQ, Hong CL (2009) Bienzymefunctionalized three-layer composite magnetic nanoparticles forelectrochemical immunosensors. Biomaterials 30(12):2284–2290

138. Zhang Y, Zeng GM, Tang L, Yu HY, Li JB (2007) Catecholbiosensor based on immobilizing laccase to modified core-shellmagnetic nanoparticles supported on carbon paste electrode.Huanjing Kexue 28(10)

139. Tang L, Zeng G, Liu J, Xu X, Zhang Y, Shen G, Li Y, Liu C (2008)Catechol determination in compost bioremediation using a laccasesensor and artificial neural networks. Anal Bioanal Chem 391(2):679–685

140. Wang S, Tan Y, Zhao D, Liu G (2008) Amperometric tyrosinasebiosensor based on Fe3O4 nanoparticles-chitosan nanocomposite.Biosens Bioelectron 23(12):1781–1787

141. Peacuterez-Loacutepez B, Merkoccedili A (2011) Magneticnanoparticles modified with carbon nanotubes for electrocatalyticmagnetoswitchable biosensing applications. Adv Funct Mater21(2):58–6363

142. Liu ZM, Liu YL, Yang HF, Yang Y, Shen GL, Yu RQ (2005) Aphenol biosensor based on immobilizing tyrosinase to modifiedcore-shell magnetic nanoparticles supported at a carbon paste elec-trode. Anal Chim Acta 533(1):3–9

143. Wu S, Wang H, Tao S, Wang C, Zhang L, Liu Z, Meng C (2011)Magnetic loading of tyrosinase- Fe3O4/mesoporous silica core/shellmicrospheres for high sensitive electrochemical biosensing. AnalChim Acta 686(1–2):81–86

144. Cevik E, Senel M, Baykal A, Abasiyanik MF (2012) A novel amper-ometric phenol biosensor based on immobilized HRP onpoly(glycidylmethacrylate)-grafted iron oxide nanoparticles for the de-termination of phenol derivatives. Sensors Actuators B 173:396–405

145. Eguilaz M, Villalonga R, Yanez-Sedeno P, Pingarron JM (2011)Designing electrochemical interfaces with functionalized magneticnanoparticles and wrapped carbon nanotubes as platforms for theconstruction of high-performance bienzyme biosensors. Anal Chem83(20):7807–7814

146. Yu DH, Blankert B, Bodoki E, Bollo S, Vire JC, Sandulescu R,Nomura A, Kauffmann JM (2006) Amperometric biosensor basedon horseradish peroxidase-immobilised magnetic microparticles.Sensors Actuators B 113(2):749–754

147. Kacar C, Erden PS, Pekyardimci S, Kilic E (2012) An Fe3O4-nanoparticles-based amperometric biosensor for creatine determina-tion. Artif Cell Nanomed Biotechnol 41(1):2–7

148. Dong XY, Mi XN, Wang B, Xu JJ, Chen HY (2011) Signal ampli-fication for DNA detection based on the HRP-functionalized Fe3O4

nanoparticles. Talanta 84(2):531–537149. Sima VH, Patris S, Aydogmus Z, Sarakbi A, Sandulescu R,

Kauffmann JM (2011) Tyrosinase immobilized magnetic

Enzymatic biosensors 19

nanobeads for the amperometric assay of enzyme inhibitors: appli-cation to the skin whitening agents. Talanta 83(3):980–987

150. Li K, Lai YJ, Zhang W, Jin LT (2011) Fe2O3@Au core/shellnanoparticle-based electrochemical DNA biosensor for escherichiacoli detection. Talanta 84(3):607–613

151. Martinez NA, Schneider RJ, Messina GA, Raba J (2010) Modifiedparamagnetic beads in a microfluidic system for the determinationof ethinylestradiol (EE2) in river water samples. BiosensBioelectron 25(6):1376–1381

152. Chawla S, Pundir CS (2011) An electrochemical biosensor forfructosyl valine for glycosylated hemoglobin detection based oncore-shell magnetic bionanoparticles modified gold electrode.Biosens Bioelectron 26(8):3438–3443

153. ZhangY, ZengGM, Tang L, HuangDL, JiangXY, ChenYN (2007)A hydroquinone biosensor using modified core-shell magneticnanoparticles supported on carbon paste electrode. BiosensBioelectron 22(9–10):2121–2126

154. Zhao YT, Zhang WY, Lin YH, Du D (2013) The vital function ofFe3O4@Au nanocomposites for hydrolase biosensor design and itsapplication in detection of methyl parathion. Nanoscale 5(3):1121–1126

155. Torres-Chavolla E, Alocilja EC (2011) Nanoparticle based DNAbiosensor for tuberculosis detection using thermophilic helicase-dependent isothermal amplification. Biosens Bioelectron 26(11):4614–4618

156. Teymourian H, Salimi A, Hallaj R (2012) Low potential detection ofNADH based on Fe3O4 nanoparticles/multiwalled carbonnanotubes composite: fabrication of integrated dehydrogenase-based lactate biosensor. Biosens Bioelectron 33(1):60–68

157. Bonel L, Vidal JC, Duato P, Castillo JR (2011) An electrochemicalcompetitive biosensor for ochratoxin a based on a DNA biotinylatedaptamer. Biosens Bioelectron 26(7):3254–3259

158. Vidal JC, Bonel L, Ezquerra A, Duato P, Castillo JR (2012) Anelectrochemical immunosensor for ochratoxin A determination inwines based on a monoclonal antibody and paramagneticmicrobeads. Anal Bioanal Chem 403(6):1585–1593

159. Min H, Qu Y-H, Li XH, Xie ZH, Wei YY, Jin LT (2007) Au-dopedFe3O4 nanoparticle immobilized acetylchblinesterase sensor for thedetection of organophosphorus pesticide. Acta Chim Sin 65(20):2303–2308

160. Gan N, Yang X, Xie DH, Wu YZ, Wen WG (2010) A disposableorganophosphorus pesticides enzyme biosensor based on magneticcomposite nano-particles modified screen printed carbon electrode.Sensors 10(1):625–638

161. Rawal R, Chawla S, Pundir CS (2012) An electrochemical sulfitebiosensor based on gold coated magnetic nanoparticles modifiedgold electrode. Biosens Bioelectron 31(1):144–150

162. Villalonga R, Villalonga ML, Diez P, Pingarron JM (2011) Deco-rating carbon nanotubes with polyethylene glycol-coated magneticnanoparticles for implementing highly sensitive enzyme biosensors.J Mater Chem 21(34):12858–12864

163. Sahraoui Y, Barhoumi H, Maaref A, Nicole JR (2011) A novelcapacitive biosensor for urea assay based on modified magneticnanobeads. Sens Lett 9(6):2141–2146

164. Nouira W, Maaref A, Elaissari H, Vocanson F, Siadat M, Jaffrezic-Renault N (2013) Comparative study of conductometric glucosebiosensor based on gold and on magnetic nanoparticles. Mater SciEng C Mater Biol Appl 33(1):298–303

165. Taton K, Johnson D, Guire P, Lange E, Tondra M (2009) Lateralflow immunoassay using magnetoresistive sensors. J Magn MagnMater 321(10):1679–1682

166. Khun K, Ibupoto ZH, Lu J, Alsalhi MS, Atif M, Ansari AA,Willander M (2012) Potentiometric glucose sensor based on theglucose oxidase immobilized iron ferrite magnetic particle/chitosancomposite modified gold coated glass electrode. Sensors ActuatorsB 173:698–703

167. Cevik E, Senel M, Baykal A (2013) Potentiometric urea biosensorbased on poly(glycidylmethacrylate)-grafted iron oxidenanoparticles. Curr Appl Phys 13(1):280–286

168. Barthelmebs L, Hayat A, Limiadi AW, Marty JL, Noguer T (2011)Electrochemical DNA aptamer-based biosensor for OTA detectionusing superparamagnetic nanoparticles. Sensors Actuators B156(2):932–937

169. Zhang ZX, Wang ZJ, Wang XL, Yang XR (2010) Magneticnanoparticle-linked colorimetric aptasensor for the detection ofthrombin. Sensors Actuators B 147(2):428–433

170. Chang Q, Deng KJ, Zhu LH, Jiang GD, Yu C, Tang HQ (2009)Determination of hydrogen peroxide with the aid of peroxidase-likeFe3O4 magnetic nanoparticles as the catalyst. Microchim Acta165(3–4):299–305

171. Kim MI, Shim J, Parab HJ, Shin SC, Lee J, Park HG (2012) Aconvenient alcohol sensor using one-pot nanocomposite entrappingalcohol oxidase andmagnetic nanoparticles as peroxidase mimetics.J Nanosci Nanotechnol 12(7):5914–5919

172. Kim MI, Shim J, Li T, Woo M-A, Cho D, Lee J, Park HG (2012)Colorimetric quantification of galactose using a nanostructuredmulti-catalyst system entrapping galactose oxidase and magneticnanoparticles as peroxidase mimetics. Analyst 137(5):1137–1143

173. Xu Q, Li JP, Li SH, Pan HC (2012) A highly sensitiveelectrochemiluminescence immunosensor based on magneticnanoparticles and its application in CA125 determination. J SolidState Electrochem 16(9):2891–2898

174. Zhou H, Gan N, Li T, Cao Y, Zeng S, Zheng L, Guo Z (2012) Thesandwich-type electrochemiluminescence immunosensor for alpha-fetoprotein based on enrichment by Fe3O4-Au magnetic nanoprobes and signal amplification byCdS-Au composite nanoparticleslabeled anti-AFP. Anal Chim Acta 746:107–113

175. Baratella D, Magro M, Siligaglia G, Zboril R, Salviulo G, VianelloF (2013) A glucose biosensor based on surface active maghemitenanoparticles. Biosens Bioelectron 45:13–18

176. Rossi LM, Quach AD, Rosenzweig Z (2004) Glucose oxidase-magnetite nanoparticle bioconjugate for glucose sensing. AnalBioanal Chem 380(4):606–613

177. Magro M, Sinigaglia G, Nodari L, Tucek J, Polakova K,Marusak Z, Cardillo S, Salviulo G, Russo U, Stevanato R,Zboril R, Vianello F (2012) Charge binding of rhodaminederivative to OH- stabilized nanomaghemite: universalnanocarrier for construction of magnetofluorescent biosen-sors. Acta Biomater 8(6):2068–2076

178. Pospiskova K, Safarik I, Sebela M, Kuncova G (2013) Magneticparticles-based biosensor for biogenic amines using an optical ox-ygen sensor as a transducer. Microchim Acta 180(3–4):311–318

179. Chang Q, Zhu L, Jiang G, Tang H (2009) Sensitive fluorescentprobes for determination of hydrogen peroxide and glucose basedon enzyme-immobilized magnetite/silica nanoparticles. AnalBioanal Chem 395(7):2377–2385

180. Wu SJ, Duan N, Wang ZP, Wang HX (2011) Aptamer-functionalized magnetic nanoparticle-based bioassay for the detec-tion of ochratoxin a using upconversion nanoparticles as labels.Analyst 136(11):2306–2314

181. Wang J, Zhu Z, Munir A, Zhou HS (2011) Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay.Talanta 84(3):783–788

182. Liang R-P, Yao G-H, Fan L-X, Qiu J-D (2012) MagneticFe3O4@Au composite-enhanced surface plasmon resonance forultrasensitive detection of magnetic nanoparticle-enriched alpha-fetoprotein. Anal Chim Acta 737:22–28

183. Shi C, GeY, GuH,MaC (2011) Highly sensitive chemiluminescentpoint mutation detection by circular strand-displacement amplifica-tion reaction. Biosens Bioelectron 26(12):4697–4701

184. Wang S, Su P, Huang J, Wu J, Yang Y (2013) Magneticnanoparticles coated with immobilized alkaline phosphatase for

20 X. Shi et al.

enzymolysis and enzyme inhibition assays. J Mater Chem B 1(12):1749–1754

185. Peng R, Zhang W, Ran Q, Liang C, Jing L, Ye S, Xian Y (2011)Magnetically switchable bioelectrocatalytic system based on ferro-cene grafted iron oxide nanoparticles. Langmuir 27(6):2910–2916

186. Liang C, Jing L, Shi X, Zhang Y, Xian Y (2012) Magneticallycontrolled bioelectrocatalytic system based on ferrocene-taggedmagnetic nanoparticles by thiol-ene reaction. Electrochim Acta 69:167–173

187. Wei H, Wang E (2008) Fe3O4 magnetic nanoparticles as peroxidasemimetics and their applications in H2O2 and glucose detection. AnalChem 80(6):2250–2254

188. LiangM, FanK, PanY, JiangH,Wang F,YangD, LuD, Feng J, ZhaoJ, Yang L, Yan X (2013) Fe3O4 magnetic nanoparticle peroxidasemimetic-based colorimetric assay for the rapid detection of organo-phosphorus pesticide and nerve agent. Anal Chem 85(1):308–312

189. Zhang ZX, Wang XL, Yang XR (2011) A sensitive choline biosen-sor using Fe3O4 magnetic nanoparticles as peroxidase mimics.Analyst 136(23):4960–4965

190. Liu BH, Cao Y, ChenDD, Kong JL, Deng JQ (2003) Amperometricbiosensor based on a nanoporous ZrO2 matrix. Anal Chim Acta478(1):59–66

191. Tong Z, Yuan R, Chai Y, Xie Y, Chen S (2007) A novel and simplebiomolecules immobilization method: electro-deposition ZrO2

doped with HRP for fabrication of hydrogen peroxide biosensor. JBiotechnol 128(3):567–575

192. Tong Z, Yuan R, Chai Y, Chen S, Xie Y (2007) Direct electrochem-istry of horseradish peroxidase immobilized on DNA/electrodeposited zirconium dioxide modified, gold disk electrode.Biotechnol Lett 29(5):791–795

193. Yang YH, Yang HF, Yang MH, Liu YL, Shen GL, Yu RQ (2004)Amperometric glucose biosensor based on a surface treatednanoporous ZrO2/chitosan composite film as immobilization ma-trix. Anal Chim Acta 525(2):213–220

194. Cai CJ, Xu MW, Bao SJ, Lei C, Jia DZ (2012) A facile route forconstructing a graphene-chitosan-ZrO2 composite for direct electrontransfer and glucose sensing. RSC Advances 2(21):8172–8178

195. Yang YH, Guo MM, Yang MH, Wang ZJ, Shen GL, Yu RQ (2005)Determination of pesticides in vegetable samples using an acetyl-cholinesterase biosensor based on nanoparticles ZrO2/chitosan com-posite film. Int J Environ Anal Chem 85(3):163–175

196. Wang H, Wang J, Choi D, Tang Z, Wu H, Lin Y (2009) EQCMimmunoassay for phosphorylated acetylcholinesterase as a biomark-er for organophosphate exposures based on selective zirconia ad-sorption and enzyme-catalytic precipitation. Biosens Bioelectron24(8):2377–2383

197. MoghaddamAB, Ganjali MR, Saboury A,Moosavi-Movahedi AA,Norouzi P (2008) Electrodeposition of nickel oxide nanoparticles onglassy carbon surfaces: application to the direct electron transfer oftyrosinase. J Appl Electrochem 38(9):1233–1239

198. Shamsipur M, Asgari M, Mousavi MF, Davarkhah R (2012) Anovel hydrogen peroxide sensor based on the direct electron transferof catalase immobilized on nano-sized NiO/MWCNTs compositefilm. Electroanalysis 24(2):357–367

199. Arora K, Tomar M, Gupta V (2011) Highly sensitive and selectiveuric acid biosensor based on rf sputtered NiO thin film. BiosensBioelectron 30(1):333–336

200. Tyagi M, Tomar M, Gupta V (2012) Influence of hole mobility onthe response characteristics of p-type nickel oxide thin film basedglucose biosensor. Anal Chim Acta 726:93–101

201. Singh J, Kalita P, Singh MK, Malhotra BD (2011) Nanostructurednickel oxide-chitosan film for application to cholesterol sensor.Appl Phys Lett 98(12):123702

202. Yadav SK, Singh J, Agrawal VV, Malhotra BD (2012) Nanostruc-tured nickel oxide film for application to fish freshness biosensor.Appl Phys Lett 101(2):023703

203. Lata S, Batra B, Karwasra N, Pundir CS (2012) An amperometricH2O2 biosensor based on cytochrome c immobilized onto nickeloxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode. Process Biochem 47(6):992–998

204. Tyagi M, Tomar M, Gupta V (2013) NiO nanoparticle-based ureabiosensor. Biosens Bioelectron 41:110–115

205. Sharifi E, Salimi A, Shams E (2013) Electrocatalytic activity ofnickel oxide nanoparticles as mediatorless system for NADH andethanol sensing at physiological pH solution. Biosens Bioelectron45:260–266

206. Yin LT, Chou JC, Chung WY, Sun TP, Hsiung KP, Hsiung SK(2001) Glucose ENFET doped with MnO2 powder. Sensors Actu-ators B 76(1–3):187–192

207. Luo XL, Xu JJ, Zhao W, Chen HY (2004) A novel glucose enfetbased on the special reactivity of MnO2 nanoparticles. BiosensBioelectron 19(10):1295–1300

208. Turkusic E, Kalcher J, Kahrovic E, Beyene NW, Moderegger H,Sofic E, Begic S, Kalcher K (2005) Amperometric determination ofbonded glucose with an MnO2 and glucose oxidase bulk-modifiedscreen-printed electrode using flow-injection analysis. Talanta65(2):559–564

209. Cui X, Liu G, Lin Y (2005) Amperometric biosensors based oncarbon paste electrodes modified with nanostructured mixed-valence manganese oxides and glucose oxidase. NanomedNanotechnol 1(2):130–135

210. Xu J-J, Feng JJ, Zhong X, Chen H-Y (2008) Low-potential detec-tion of glucose with a biosensor based on the immobilization ofglucose oxidase on polymer/manganese oxide layered nanocom-posite. Electroanalysis 20(5):507–512

211. Yoshimoto M, Iida C, Kariya A, Takaki N, Nakayama M (2010) Abiosensor composed of glucose oxidase-containing liposomes andMnO2-based layered nanocomposite. Electroanalysis 22(6):653–659

212. Yang X, Chen X, Zhang X, YangW, Evans DG (2008) Intercalationof methylene blue into layered manganese oxide and application ofthe resulting material in a reagentless hydrogen peroxide biosensor.Sensors Actuators B 129(2):784–789

213. Xiushuang Y, Xu C, Xiong Z, Wensheng Y, Evans DG (2008)Direct electrochemistry and electrocatalysis with horseradish per-oxidase immobilized in polyquaternium-manganese oxidenanosheet nanocomposite films. Sensors Actuators B 134(1):182–188

214. Mohseni G, Negahdary M, Faramarzi H, Mehrtashfar S, Habibi-Tamijani A, Nazemi SH,Morshedtalab Z,MazdapourM, Parsania S(2012) Voltammetry behavior of modified carbon paste electrodewith cytochrome c and Mn2O3 nanoparticles for hydrogen peroxidesensing. Int J Electrochem Sci 7(12):12098–12109

215. Telsnig D, Kalcher K, Leitner A, Ortner A (2013) Design of anamperometric biosensor for the determination of biogenic aminesusing screen printed carbon working electrodes. Electroanalysis25(1):47–50

216. Liu ZJ, Liu BH, Kong JL, Deng JQ (2000) Probing trace phenolsbased on mediator-free alumina sol-gel derived tyrosinase biosen-sor. Anal Chem 72(19):4707–4712

217. Gao ZQ, Xie F, Shariff M, Arshad M, Ying JY (2005) A disposableglucose biosensor based on diffusional mediator dispersed innanoparticulate membrane on screen-printed carbon electrode. Sen-sors Actuators B 111:339–346

218. Darder M, Aranda P, Hernandez VM, Manova E, Ruiz HE (2006)Encapsulation of enzymes in aluminamembranes of controlled poresize. Thin Solid Films 495(1–2):321–326

219. Shi MH, Xu JJ, Zhang S, Liu BH, Kong JL (2006) A mediator-freescreen-printed amperometric biosensor for screening of organo-phosphorus pesticides with flow-injection analysis (FIA) system.Talanta 68(4):1089–1095

Enzymatic biosensors 21

220. Wu F, Hu Z, Wang L, Xu J, Xian Y, Tian Y, Jin L (2008) Electricfield directed layer-by-layer assembly of horseradish peroxidasenanotubes via anodic aluminum oxide template. ElectrochemCommun 10(4):630–634

221. Liu X, Luo L, Ding Y, Xu Y (2011) Amperometric biosensors basedon alumina nanoparticles-chitosan-horseradish peroxidasenanobiocomposites for the determination of phenolic compounds.Analyst 136(4):696–701

222. Ansari SA, Husain Q (2011) Immobilization of kluyveromyceslactis beta galactosidase on concanavalin a layered aluminium oxidenanoparticles-its future aspects in biosensor applications. J MolCatal B 70(3–4):119–126

223. Santos A, Macias G, Ferre-Borrull J, Pallares J, Marsal LF (2012)Photoluminescent enzymatic sensor based on nanoporous anodicalumina. ACS Appl Mater Interfaces 4(7):3584–3588

224. Li Y,Wei Y, Shi G, Xian Y, Jin L (2011) Facile synthesis of leaf-likecuo nanoparticles and their application on glucose biosensor. Elec-troanalysis 23(2):497–502

225. Ding SN, Shan D, Xue HG, Cosnier S (2010) A promisingbiosensing-platform based on bismuth oxide polycrystalline-modified electrode: characterization and its application in develop-ment of amperometric glucose sensor. Bioelectrochemistry 79(2):218–222

226. Saha S, Arya SK, Singh SP, Sreenivas K, Malhotra BD, Gupta V(2009) Nanoporous cerium oxide thin film for glucose biosensor.Biosens Bioelectron 24(7):2040–2045

227. Zhao JW, Qin LR, Hao YH, Guo Q, Mu F, Yan ZK (2012) Appli-cation of tubular tetrapod magnesium oxide in a biosensor forhydrogen peroxide. Microchim Acta 178(3–4):439–445

228. Periasamy AP, Ting SW, Chen SM (2011) Amperometric andimpedimetric H2O2 biosensor based on horseradish peroxidasecovalently immobilized at ruthenium oxide nanoparticles modifiedelectrode. Int J Electron Sc 6(7):2688–2709

229. Lavanya N, Radhakrishnan S, Sekar C (2012) Fabrication of hy-drogen peroxide biosensor based on Ni doped SnO2 nanoparticles.Biosens Bioelectron 36(1):41–47

230. Wang J, Yuan R, Chai Y, Li W, Fu P, Min L (2010) Usingflowerlike polymer-copper nanostructure composite and novelorganic-inorganic hybrid material to construct an amperomet-ric biosensor for hydrogen peroxide. Collods Surf B 75(2):425–431

231. Zong S, Cao Y, Zhou Y, Ju H (2007) Hydrogen peroxide biosensorbased on hemoglobin modified zirconia nanoparticles-grafted col-lagen matrix. Anal Chim Acta 582(2):361–366

232. Salimi A, Sharifi E, Noorbakhsh A, Soltanian S (2007) Directelectrochemistry and electrocatalytic activity of catalaseimmobilized onto electrodeposited nano-scale islands of nickeloxide. Biophys Chem 125(2–3):540–548

233. Qu S, Pei SP, Zhou SL, Gu YY (2009) One-step electrodepositedcarbon nanotube/zirconia/myoglobin film for direct electron transferand electrocatalysis. J Chin Chem Soc 56(4):822–827

234. Zong SZ, Cao Y, Zhou YM, Ju HX (2006) Zirconiananoparticles enhanced grafted collagen tri-helix scaffoldfor unmediated biosensing of hydrogen peroxide. Langmuir22(21):8915–8919

235. Bai YH, DuY, Xu JJ, Chen HY (2007) Choline biosensors based ona bi-electrocatalytic property of MnO2 nanoparticles modified elec-trodes to H2O2. Electrochem Commun 9(10):2611–2616

236. Chen DD, Liu BH, Liu ZJ, Kong JL (2001) An amperometricbiosensor for hydrogen peroxidase based on the co-immobilization of catalase and methylene blue in an Al2O3 sol-gelmodified electrode. Anal Lett 34(5):687–699

237. Chen DD, Liu BH, Kong JL (2002) A hydrogen peroxide biosensorbased on Al2O3 sol-gel immobilizing horseradish peroxidase andthionine. Chin J Anal Chem 30(8):958–961

238. Periasarny AP, Yang SY, Chen SM (2011) Preparation and charac-terization of bismuth oxide nanoparticles-multiwalled carbon nano-tube composite for the development of horseradish peroxidasebased H2O2 biosensor. Talanta 87:15–23

239. Salimi A, Sharifi E, Noorbakhsh A, Soltanian S (2007) Immobili-zation of glucose oxidase on electrodeposited nickel oxidenanoparticles: Direct electron transfer and electrocatalytic activity.Biosens Bioelectron 22(12):3146–3153

240. Yu JJ, Zhao T, Zeng BZ (2008) Mesoporous MnO2 as enzymeimmobilization host for amperometric glucose biosensor construc-tion. Electrochem Commun 10(9):1318–1321

241. Liu ZJ, Liu BH, Zhang M, Kong JL, Deng JQ (1999) Al2O3 sol-gelderived amperometric biosensor for glucose. Anal Chim Acta392(2–3):135–141

242. Lin C, Xiong ZX, Xue H, Chen SX, Qiu H (2011) Amperometricbiosensor with Al2O3/Al foil electrodes modified by Pt nanofuzz forglucose detection. Sensors Mater 23(5):293–302

243. Patil D, Dung NQ, Jung H, Ahn SY, Jang DM, Kim D (2012)Enzymatic glucose biosensor based on CeO2 nanorods synthesizedby non-isothermal precipitation. Biosens Bioelectron 31(1):176–181

244. Xu JJ, Zhao W, Luo XL, Chen HY (2005) A sensitive biosensor forlactate based on layer-by-layer assembling MnO2 nanoparticles andlactate oxidase on ion-sensitive field-effect transistors. ChemCommun 41(6):792–794

245. Wang K, Xu JJ, Chen HY (2006) Biocomposite of cobalt phthalo-cyanine and lactate oxidase for lactate biosensing with MnO2

nanoparticles as an eliminator of ascorbic acid interference. SensorsActuators B 114(2):1052–1058

246. Batra B, Lata S, Sunny RJS, Pundir CS (2013) Construction of anamperometric bilirubin biosensor based on covalent immobilizationof bilirubin oxidase onto zirconia coated silica nanoparticles/chitosan hybrid film. Biosens Bioelectron 44:64–69

247. Singh J, Srivastava M, Kalita P, Malhotra BD (2012) A novelternary NiFe2O4/CuO/FeO-chitosan nanocomposite as a cholesterolbiosensor. Process Biochem 47(12):2189–2198

248. Batra B, Lata S, Rani S, Pundir CS (2013) Fabrication of a cytochromec biosensor based on cytochrome oxidase/NiO-NPs/CMWCNT/PANImodified Au electrode. J Biomed Nanotechnol 9(3):409–416

249. Aydogdu G, Zeybek DK, Zeybek B, Pekyardimci S (2013) Electro-chemical sensing of NADH on NiO nanoparticles-modified carbonpaste electrode and fabrication of ethanol dehydrogenase-basedbiosensor. J Appl Electrochem 43(5):523–531

250. Rawal R, Chawla S, Malik P, Pundir CS (2012) An amperometricbiosensor based on laccase immobilized onto MnO(2)NPs/CMWCNT/PANI modified au electrode. Int J Biol Macromol51(1–2):175–181

251. Zejli H, De Cisneros J, Naranjo-Rodriguez I, Liu BH, TemsamaniKR, Marty JL (2008) Phenol biosensor based on sonogel-carbontransducer with tyrosinase alumina sol-gel immobilization. AnalChim Acta 612(2):198–203

252. Wan Y, Qi P, Zhang D, Wu JJ, Wang Y (2012) Manganese oxidenanowire-mediated enzyme-linked immunosorbent assay. BiosensBioelectron 33(1):69–74

253. Yang ZP, Si SH, Dai HJ, Zhang CJ (2007) Piezoelectric ureabiosensor based on immobilization of urease onto nanoporousalumina membranes. Biosens Bioelectron 22(12):3283–3287

254. Jindal K, Tomar M, Gupta V (2012) CuO thin film based uric acidbiosensor with enhanced response characteristics. BiosensBioelectron 38(1):11–18

255. Luong JHT, Male KB, Glennon JD (2008) Biosensor technology:technology push versus market pull. Biotechnol Adv 26(5):492–500

256. Karel H, Fernandez-Lafuente R (2011) Control of protein immobi-lization: coupling immobilization and site-directed mutagenesis toimprove biocatalyst or biosensor performance. Enzyme MicrobTechnol 48:107–122

22 X. Shi et al.