Hydrogen peroxide biosensor based on horseradish ... - Springer Link

2 downloads 0 Views 293KB Size Report
Apr 8, 2011 - Abstract A biosensor for hydrogen peroxide was con- structed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe2O4 ...
Microchim Acta (2011) 174:55–61 DOI 10.1007/s00604-011-0591-6

ORIGINAL PAPER

Hydrogen peroxide biosensor based on horseradish peroxidase immobilized on chitosan-wrapped NiFe2O4 nanoparticles Liqiang Luo & Limei Zhu & Yanhong Xu & Liuyi Shen & Xia Wang & Yaping Ding & Qiuxia Li & Dongmei Deng

Received: 26 December 2010 / Accepted: 21 March 2011 / Published online: 8 April 2011 # Springer-Verlag 2011

Abstract A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe2O4 nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV–vis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H2O2 in the range from 1.0×10−5 to 2.0×10−3 M and with a detection limit of 2.0×10−6 M (at S/N=3). Keywords Chitosan . NiFe2O4 nanoparticles . Horseradish peroxidase . Ferrocene carboxylic acid . Biosensor

Introduction Spinel ferrite nanoparticles have been investigated intensively in recent years because of their remarkable electrical and magnetic properties and wide practical applications for the development of immunosensors, biosensors and bioelectronic devices [1–5]. The super paramagnetic and ferrimagnetic nanoparticles provide attractive biotechnical and physiological advantages: (1) direct injection (or

L. Luo (*) : L. Zhu : Y. Xu : L. Shen : X. Wang : Y. Ding (*) : Q. Li : D. Deng College of Sciences, Shanghai University, Shanghai 200444, People’s Republic of China e-mail: [email protected] Y. Ding e-mail: [email protected]

registration) through blood vessel due to easy controlling of particle size, (2) remote controlling of transport through blood vessels to the targeted cell (tumor cell) by externally applied magnetic field gradients, (3) possibility for the differentiation of tumor cells from healthy cells by using antigen-antibody biological reactions and (4) immobilization for bioelectronic applications [6–10]. Chitosan (CHIT), a copolymer of glucosamine and Nacetyglucosamine units linked by 1–4 glucosidic bonds, can be obtained by N-deacetylation of chitin, which is the second most abundant natural polymer [11]. In recent years, CHIT has attracted much attention owing to its specific properties such as biodegradability, biocompatibility, physiological inertness and high mechanical strength [12, 13]. In addition, CHIT has the susceptibility to chemical modifications due to the abundant amino groups and it can form a thermally and chemically inert film that is insoluble in water [14]. So, CHIT has been widely used in the preparation of sensors in recent years [3, 15–21]. A key challenge of developing enzyme-based biosensors is to improve the electron transfer between the electrode and the redox enzyme. Slow dynamics frequently arising from the enzyme’s redox center is encapsulated. A highly successful approach to increasing the electron-transfer rate is to “electrochemically wire” the enzyme by either attaching redox-active mediators which have a suitable redox potential, directly to the enzyme or using a redoxconducting polymer that can shuttle electrons to and from the active site [22–24]. Because of their good stability in solution, rapid responses to many electroactive substances, stability in both oxidized and reduced forms, unreactivity with oxygen, regeneration at low potential and having fast electron transfer, ferrocene and its derivatives are the most widely used class of mediators in the fabrication of stable and sensitive biosensors [25–30].

56

Owing to unique properties such as large surface-tovolume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability, applications of nanomaterials to biosensors have recently aroused much interest [31]. Recently, we have verified that the NiFe2O4 nanoparticles (NiFe2O4NPs)/CHIT composite film shows an excellent electrocatalytical response to the oxidation of glucose [32]. In order to extend the applications of this composite film, herein we fabricated a hydrogen peroxide (H2O2) biosensor based on the immobilization of horseradish peroxidase in the film mentioned above. Furthermore, ferrocene carboxylic acid (Fc), which was used as a mediator, was immbolized on the surface of electrode to make the biosensor better practical. NiFe2O4NPs with inverse spinel structure show good biocompatibility, noncytotoxicity and easy preparation process [33], thus, it is chosen to immobilize horseradish peroxidase (HRP) in the matrix. This modified glassy carbon electrode (GCE) shows efficient catalytic activity toward H2O2.

Experimental

L. Luo et al.

CHIT powder in 0.1 M acetic acid solution. 2 mg of NiFe2O4NPs were suspended in 1.0 mL of CHIT solution. The mixture was placed for 3 h in an ultrasonic bath in order to keep the suspension homogeneous. Then certain amount of HRP was dissolved in NiFe2O4NPs/CHIT suspension. 5 μL of Fc solution (100 mM) was pipetted onto the surface of the electrode and dried in atmosphere. 8 μL of HRP/NiFe2O4NPs/CHIT suspension was dropped onto the surface of the GCE secondly, and distributed gently over the entire surface to ensure the complete coating of the GCE by the HRP solution. Then, the electrode was left to dry at 4 °C. Before utilization, the electrode was soaked into 0.1 M phosphate buffer solution (pH 7.0) at room temperature for 2 h, to restore the enzyme activity. When not in use, the biosensor was stored at 4 °C in 0.1 M phosphate buffer solution. Apparatus and measurements Electrochemical experiments were performed on a CHI 842B electrochemical station (CH Instruments, China) with a conventional three-electrode system. The GCE with modified film acted as the working electrode. A saturated

Materials and reagents HRP (300 U mg−1) was purchased from Dingguo Bioengineering Company (Beijing, http://www.dingguo.com). Fc was purchased from Sigma. USA. (http://www.sigmaaldrich. com) H2O2 (30%), KH2PO4, K2HPO4·3H2O, K3[Fe(CN)6], K4[Fe(CN)6], KCl, NiSO4·6H2O, FeSO4·7H2O, Fe2(SO4)3 and CHIT were bought from Sinopharm Chemical Reagent Co. Ltd. (http://shreagent.en.alibaba.com). 0.1 M phosphate buffer solution was used as the supporting electrolyte. All chemicals were analytical grade. All solutions were made with double distilled water. Preparation of HRP/NiFe2O4NPs/CHIT/Fc/GCE The NiFe2O4NPs were prepared according to the literature [34]. Briefly, 0.3 M NiSO4·6H2O, 0.5 M FeSO4·7H2O and 0.1 M Fe2(SO4)3 were dissolved in N2-saturated deionized water. The solution of NaOH (1.5 M) was added dropwise to the stirred mixture until pH 7.0. The mixture was filtered and washed with N2-saturated deionized water and ethanol. A pure ferrite spinel, NiFe2O4NPs, was obtained by calcination of the layered double hydroxide precursor containing Ni2+, Fe2+ and Fe3+ cations, which was proved by X-ray diffraction [6]. GCE (i.d.=3 mm) was polished before each experiment with 0.05 μm alumina powder successively rinsed thoroughly with absolute alcohol and distilled water in ultrasonic bath and dried in air. CHIT solution (0.5%) was prepared by dissolving

Fig. 1 a UV–vis absorption spectra of (a) HRP and (b) HRP/ NiFe2O4NPs/CHIT/Fc on quartz plates; b FTIR transmission spectra of (a) HRP and (b) HRP/NiFe2O4NPs/CHIT/Fc

Hydrogen peroxide biosensor based on horseradish peroxidase Fig. 2 SEM images of a Fc and b HRP/NiFe2O4NPs/CHIT/Fc on glassy carbon plates

a

57

b

calomel electrode (SCE) was used as the reference electrode and a platinum foil acted as the auxiliary electrode. Scanning electron microscopic (SEM) measurements were carried out on a scanning electron microscope (JEOL JSM-6700F) at 20 kV. Electrochemical impedance spectra (EIS) were performed in 5 mM Fe(CN)63−/4− containing 0.1 M KCl on Solartron 1255B Frequency Response analyzer/SI 1287 electrochemical interface (Scribner Associates, Inc.). The frequency scan range was from 10−1 to 105 Hz and the amplitude was 10 mV. UV–vis spectra were performed on UV-2501 and fourier transform infrared (FTIR) were carried out on AVATAR 370.

NiFe2O4NPs/CHIT/Fc composite film are nearly the same as those obtained for native HRP film, which correspond to C=O stretching vibration of peptide linkages and N-H bending and C-N stretching vibration in the backbone of protein [35]. FTIR spectroscopy also proves that the structure of HRP kept almost unchanged in HRP/ NiFe2O4NPs/CHIT/Fc film. The morphology of HRP/NiFe2O4NPs/CHIT/Fc film was characterized by SEM. Figure 2 shows the SEM images of Fc (a) and HRP/NiFe2O4NPs/CHIT/Fc (b) on glassy carbon plates. As can be seen in Fig. 2, Fc was crystallized on the matrix and HRP/NiFe2O4NPs/CHIT exhibited island-like structures, which was due to the presence of NiFe2O4NPs. Such network-like structure increases the effective surface area of the substrate electrode to load large amount of HRP.

Results and discussion

Electrochemical characterization of HRP/NiFe2O4NPs/ CHIT/Fc modified GCE

Spectroscopic analysis and SEM of HRP/NiFe2O4NPs/ CHIT/Fc hybrid film UV–vis analysis was used to probe the structure change for heme proteins because the location of the absorption band of iron provides structural information about possible denaturation of hemeproteins, especially conformational change in the heme group region [16]. UV–vis spectra of HRP film (curve a) and HRP/NiFe2O4NPs/ CHIT/Fc film (curve b) coating on quartz plates gave Soret bands at 406 nm (Fig. 1a). The result indicates that HRP entrapped in HRP/NiFe2O4NPs/CHIT/Fc film had a structure similar to native state of HRP in dry films and the film based on NiFe2O4NPs/CHIT/Fc restored HRP activity. FTIR spectroscopy is another effective way to investigate the structure of proteins. The FTIR spectra of HRP (curve a), HRP/NiFe2O4NPs/CHIT/Fc film (curve b) were shown in Fig. 1b. In the spectrogram, the peaks of amide I and II bands (1656 and 1,546 cm−1) of HRP in HRP/

EIS is a useful tool to compare the impedance of electrodes, with which the interface properties of surface modified

Fig. 3 EIS of a GCE, b NiFe2O4NPs/CHIT/Fc/GCE and c HRP/ NiFe2O4NPs/CHIT/Fc/GCE in 5 mM Fe(CN)63−/4− +0.1 M KCl solution. The frequency range was from 1×10−1 to 1×105 Hz

58

L. Luo et al.

Fig. 4 a The amperometric response of the biosensor to 0.2 mM H2O2 in 0.1 M phosphate buffer solution (pH 7.0) with different volume of Fc and 8 8 mg mL−1 HRP modified GCE, working potential at −0.1 V vs. SCE; b Effect of HRP concentration on the amperometric response of the biosensor to 0.2 mM H2O2 in 0.1 M phosphate buffer solution (pH 7.0), 5 μL of Fc solution modified GCE, working potential at −0.1 V vs. SCE; c Effect of working

potential on the amperometric response of the biosensor to 0.2 mM H2O2 in 0.1 M phosphate buffer solution (pH 7.0), 5 μL of Fc solution and 8 mg mL−1 HRP modified GCE; d Effect of pH on the amperometric response of the biosensor to 0.2 mM H2O2 in 0.1 M phosphate buffer solution, 5 μL of Fc solution and 8 mg mL−1 HRP modified GCE, working potential at −0.1 V vs. SCE

electrode can be studied. It is well known that the high frequency region of the impedance plot shows a semicircle related to the redox probe Fe(CN)63−/4−, followed by a Warburg line in the low frequency region which corresponds to the diffusion step of the overall process. Figure 3 presents the representative impedance spectrum of the bare GCE (A), NiFe2O4NPs/CHIT/Fc/GCE (B), and HRP/ NiFe2O4NPs/CHIT/Fc/GCE (C). The Ret value at the bare GCE was estimated to be 1,000 Ω (Fig. 3a). When NiFe2O4NPs/CHIT/Fc and HRP/NiFe2O4NPs/CHIT/Fc were modified on the bare GCE, respectively, their Ret values declined (140 Ω Fig. 3b and 200 Ω Fig. 3c). As shown in Fig. 3, compared with A, the resistances for B were decreased. The reason might be the fact that immobile Fc and NiFe2O4NPs have high charge transfer efficiency and facilitate the electrolyte to penetrate the film layer. The Ret value increase in Fig. 3c indicated the immobilized of HRP.

volume of 100 mM Fc solution. In Fig. 4a, the biosensor response to H2O2 increased sharply with the increase of Fc volume from 0 to 5 μL. Typically, when the mediator volume was small, the biosensor response was limited by

Optimization of analytical condition As an electron mediator, the effect of Fc quantity on the biosensor response was investigated by varying the

Fig. 5 Typical current—time response curves of HRP/NiFe2O4NPs/ CHIT/Fc/GCE for the successive addition of H2O2 in 0.1 M phosphate buffer solution (pH 7.0), working potential at −0.1 V vs. SCE

Hydrogen peroxide biosensor based on horseradish peroxidase

59

Table 1 Figures of merits of comparable methods for determination of H2O2 Methods

Linear range

Clay-HRP-chitosan-AuNPs modified GCE Red blood cells-Fe3O4 core/Au-cysteamine modified gold electrode HRP/DNA/L-cys/Au NPs/PPy/gold electrode HRP/NiFe2O4NPs/CHIT/Fc/GCE

39 9.6 4.9 10

μM–3.1 μM–2.6 μM–4.8 μM–2.0

mM mM mM, mM

LOD

Ref.

9.0 μM 4.40 μM 1.3 μM 2.0 μM

38 39 40 This work

the enzyme-mediator kinetics. Further increase of the volume of mediator resulted in a slight decrease in current response as the response signal was limited by the enzyme-substrate kinetics [14]. Thus, the volume of Fc solution (100 mM) was selected at 5 μL for all the subsequent experiments. To test the optimal concentration of HRP for H2O2 determination, a range of HRP concentration from 5 to 10 mg mL−1 was used in the reaction system. The result shows that the amperometric response of the biosensor to H2O2 was increased along with the increase of HRP concentration from 5 to 8 mg mL−1. When the concentration of HRP reached 8 mg/mL, the current decreased a little (Fig. 4b). Therefore, 8 mg mL−1 was elected as optimal concentration of HRP for the following experiments. The effect of applied potential on the biosensor response was also tested and the results are shown in Fig. 4c. The biosensor response to H2O2 increased with the change of applied potential from −0.4 to −0.1 V. The highest sensitivity was obtained at −0.1 V and further increase of the anodic potential resulted in decrease in current response. The applied potential was selected at −0.1 V in amperometric detection. In addition, the influence of pH of the supporting electrolyte over the range 5.5–8.5 on the amperometric response of the biosensor to 0.2 mM H2O2 in 0.1 M phosphate buffer solution was investigated. The results indicates the optimum pH was 7.0 (Fig. 4d), which was accordant to the previous literatures [36, 37].

with other methods as shown in Table 1. The detection limit was 2.0×10−6 M (3σ), which was lower than 9 μM of ClayHRP-chitosan-AuNPs modified GCE [38] and 4.40 μM of Red blood cells-Fe3O4 core/Au-cysteamine modified gold electrode [39].

Amperometric response of the biosensor

Conclusions

Figure 5 displays typical chronoamperometry of the H2O2 biosensor for successive additions of the same amounts of H2O2 under optimized experimental conditions. The biosensor exhibited a rapid and sensitive response to the change of H2O2 concentration, which indicated the nice electrocatalytic behavior of HRP/NiFe2O4NPs/CHIT/Fc/GCE. The response time is less than 5 s. The response curve for H2O2 showed a linear range from 10 μM to 2.0 mM, the regression equation was I ð2AÞ ¼ 0:2596 c ðmMÞ þ 0:8389 with R2 =0.9875. The performance of the proposed method was compared

A biocompatible film based on CHIT, NiFe2O4NPs and Fc was prepared and used for HRP assembly and biosensor fabrication. The activities of HRP were investigated by spectroscopic and electrochemical analysis. The results demonstrated that HRP retained its native secondary structure in the NiFe2O4NPs/CHIT film and bioeletrocatalytic activity with high sensitivity and fast response toward hydrogen peroxide. The result described herein is a promising approach towards sensitive enzyme-based biosensors.

Interferences and stability The interference tests were carried out in 0.1 M phosphate buffer solution containing 0.2 mM H2O2 in the presence of the same concentration of serine, L-tryptophan, L-tyrosine, L-cysteine, glucose and ascorbic acid. It was found that these substances did not cause any observable interference in the design concentration of H2O2 except ascorbic acid. Ascorbic acid can reduce the (C5H5−)Fe3+ produced in the peroxidase catalyzed reaction and thus, interferes in the determination of H2O2 [14]. The repeatability was evaluated by detecting the amperometric response to 5.0×10−5 H2O2 in 0.1 M phosphate buffer solution. The relative standard deviation (RSD) for nine replicate measurements was 4.3%. The long-term stability of this biosensor was also investigated. The HRP/ NiFe2O4NPs/CHIT/Fc electrode was stored in 0.1 M phosphate buffer solution (pH 7.0) at 4 °C when not being in use. Measured by chronoamperometry 3 days later, the current had no changes, but it was 0.85 times of the primal value a week later. In conclusion, the modified electrodes have a better stability.

60 Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 10804067, 20975066), Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50102), and the State Key Laboratory of Electroanalytical Chemistry, China (No. 2009005).

L. Luo et al.

18.

19.

References 1. Zhuo Y, Yuan PX, Yuan R, Chai YQ, Hong CL (2009) Bienzyme functionalized three-layer composite magnetic nanoparticles for electrochemical immunosensors. Biomaterials 30:2284–2290 2. Fu XH (2008) Magnetic-controlled non-competitive enzymelinked voltammetric immunoassay for carcinoembryonic antigen. Biochem Eng J 39:267–275 3. Kaushik A, Khan R, Solanki PR, Pandey P, Alam J, Ahmad S, Malhotra BD (2008) Iron oxide nanoparticles–chitosan composite based glucose biosensor. Biosens Bioelectron 24:676–683 4. Zhang LH, Zhai YM, Gao N, Wen D, Dong SJ (2008) Sensing H2O2 with layer-by-layer assembled Fe3O4–PDDA nanocomposite film. Electrochem Commun 10:1524–1526 5. Zhuang J, Zhang JB, Gao LZ, Zhang Y, Gu N, Feng J, Yang DL, Yan XY (2008) A novel application of iron oxide nanoparticles for detection of hydrogen peroxide in acid rain. Mater Lett 62:3972–3974 6. Zhang DG, Tong ZW, Xu GY, Li SZ, Ma JJ (2009) Templated fabrication of NiFe2O4 nanorods: characterization, magnetic and electrochemical properties. Solid State Sci 11:113–117 7. Kim JW, Choi SH, Lillehei PT, Chu SH, King GC, Watt GD (2007) Electrochemically controlled reconstitution of immobilized ferritins for bioelectronic applications. J Electroanal Chem 601:8– 16 8. Fuertes AB, Valdés-Solís T, Sevilla M (2008) Fabrication of monodisperse mesoporous carbon capsules decorated with ferrite nanoparticles. J Phys Chem C 112:3648–3654 9. Yu D, Blankert B, Bodoki E, Bollo S, Vire JC, Sandulescu R, Nomura A, Kauffmann JM (2008) Amperometric biosensor based on horseradish peroxidase-immobilised magnetic microparticles. Sens Actuators B 129:497–503 10. Lai GS, Zhang HL, Han DY (2008) A novel hydrogen peroxide biosensor based on hemoglobin immobilized on magnetic chitosan microspheres modified electrode. Sens Actuators B 113:749– 754 11. Wan Y, Creber KAM, Peppley B, Bui VT (2003) Characterization and ionic conductive properties of phosphorylated chitosan membranes. Macromol Chem Phys 204:850–858 12. Feng JJ, Zhao G, Xu JJ, Chen HY (2005) Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan. Anal Biochem 342:280–286 13. Sun X, Wang XY, Zhao WP (2010) Multiwall carbon nanotubebased acetylcholinesterase biosensor for detecting organophosphorous pesticides. Sens Lett 8:247–252 14. Wang HS, Pan QX, Wang GX (2005) A biosensor based on immobilization of horseradish peroxidase in chitosan matrix crosslinked with glyoxal for amperometric determination of hydrogen peroxide. Sensors 5:266–276 15. Qiu JD, Xie HY, Liang RP (2008) Preparation of porous chitosan/ carbon nanotubes film modified electrode for biosensor application. Microchim Acta 162:57–64 16. Vasilieva TM, Mahir AH, Vasiliev MN (2008) The electron beam plasma treatment–the novel approach to the controllable modification of the proteins and polysaccharides bioactivity. Sens Lett 6:496–501 17. Yang YH, Yang GM, Huang Y, Bai HP, Lu XX (2009) A new hydrogen peroxide biosensor based on gold nanoelectrode

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

35. 36.

ensembles/multiwalled carbon nanotubes/chitosan film-modified electrode. Colloids Surf A 340:50–55 Wang XL, Yang T, Feng YY, Jiao K, Li GC (2009) A novel hydrogen peroxide biosensor based on the synergistic effect of gold-platinum alloy nanoparticles/polyaniline nanotube/chitosan nanocomposite membrane. Electroanalysis 21:819–825 Kafi AKM, Chen AC (2009) A novel amperometric biosensor for the detection of nitrophenol. Talanta 79:97–102 Yang YH, Yang MH, Jiang JH, Shen GL, Yu RQ (2005) A novel biomolecular immobilization matrix based on nanoporous ZnO/ chitosan composite film for amperometric hydrogen peroxide biosensor. Chin Chem Lett 16:951–954 Wu H, Wang J, Kang X, Wang C, Wang D, Liu J, Aksay IA, Lin Y (2009) Glucose biosensor based on immobilization of glucose oxidase in platinum nanoparticles/graphene/chitosan nanocomposite film. Talanta 80:403–406 Carolan N, Forster RJ, Ó’Fágáin C (2007) Covalent attachment of ferrocene to soybean peroxidase glycans: electron transfer mediation to redox enzymes. Bioconjug Chem 18:524–529 Ammam M, Fransaer J (2010) Micro-biofuel cell powered by glucose/O2 based on electro-deposition of enzyme, conducting polymer and redox mediators: preparation, characterization and performance in human serum. Biosens Bioelectron 25:1474–1480 Bourigua S, Hafaid I, Korri-Youssoufi H, Maaref A, JaffrezicRenault N (2009) A novel urea biosensor based on modified electrodes with urease immobilized on poly(N–hydroxyphtalimide– pyrrole–co–pyrrole) film incorporating ethyl amine ferrocene as redox marker. Sens Lett 7:731–738 Raoof JB, Ojani R, Kiani A (2001) Carbon paste electrode spiked with ferrocene carboxylic acid and its application to the electrocatalytic determination of ascorbic acid. J Electroanal Chem 515:45–51 Sardar R, Beasley CA, Murray RW (2009) Ferrocenated Au nanoparticle monolayer adsorption on self–assembled monolayercoated electrodes. Anal Chem 81:6960–6965 Mehmet S, Emre C, Fatih Abasıyanık M (2010) Amperometric hydrogen peroxide biosensor based on covalent immobilization of horseradish peroxidase on ferrocene containing polymeric mediator. Sens Actuators B 145:444–450 Qiu JD, Zhou WM, Guo J, Wang R, Liang RP (2009) Amperometric sensor based on ferrocene-modified multiwalled carbon nanotube nanocomposites as electron mediator for the determination of glucose. Anal Biochem 385:264–269 Nagarale RK, Lee JM, Shin W (2009) Electrochemical properties of ferrocene modified polysiloxane/chitosan nanocomposite and its application to glucose sensor. Electrochim Acta 54:6508–6514 Wang HS, Pan QX, Wang GX (2005) Hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase in chitosan/polyvinylpyrrolidone hybrid film. Chin J Anal Chem 33:1623–1626 Wang F, Hu S (2009) Electrochemical sensors based on metal and semiconductor nanoparticles. Microchim Acta 165:1–22 Luo LQ, Li QX, Xu YH, Ding YP, Wang X, Deng DM, Xu YJ (2010) Amperometric glucose biosensor based on NiFe2O4 nanoparticles and chitosan. Sens Actuators B 145:293–298 Bae S, Lee SW, Takemura Y (2006) Applications of NiFe2O4 nanoparticles for a hyperthermia agent in biomedicine. Appl Phys Lett 89:252503/1–252503/3 Liu JJ, Li F, Evans DG, Duan X (2003) Stoichiometric synthesis of a pure ferrite from a tailored layered double hydroxide (hydrotalcite-like) precursor. Chem Commun 4:542–543 Rahmelow K, Hubner W, Ackermann T (1998) Infrared absorbances of protein side chains. Anal Biochem 257:1–11 Chang Q, Zhu LH, Jiang GD, Tang HQ (2009) Sensitive fluorescent probes for determination of hydrogen peroxide and

Hydrogen peroxide biosensor based on horseradish peroxidase glucose based on enzyme-immobilized magnetite/silica nanoparticles. Anal Bioanal Chem 395:2377–2385 37. Ansari AA, Sumana G, Khan R, Malhotra BD (2009) Polyanilinecerium oxide nanocomposite for hydrogen peroxide sensor. J Nanosci Nanotech 9:4679–4685 38. Zhao XJ, Mai ZB, Kang XH, Zou XY (2008) Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay–chitosan-gold nanoparticle nanocomposite. Biosens Bioelectron 23:1032–1038

61 39. Chen C, Liu Y, Gu HY (2010) Cellular biosensor based on red blood cells immobilized on Fe3O4 Core/Au Shell nanoparticles for hydrogen peroxide electroanalysis. Microchim Acta 171:371–376 40. Che X, Yuan R, Chai Y, Ma L, Li W, Li J (2009) Hydrogen peroxide sensor based on horseradish peroxidase immobilized on an electrode modified with DNA-L-cysteine-gold-platinum nanoparticles in polypyrrole film. Microchim Acta 167: 159–165