Journal of Electroanalytical Chemistry 624 (2008) 79–83

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

One-step biomimetic coprecipitation method to form calcium phosphate andhemoglobin composite nanoparticles for biosensing application

Hui Zhang, Jing-Juan Xu, Hong-Yuan Chen *

The Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 April 2008Received in revised form 23 July 2008Accepted 28 July 2008Available online 5 August 2008

Keywords:Biomimetic coprecipitationCalcium phosphateHemoglobinBiosensor

0022-0728/$ - see front matter � 2008 Elsevier B.V. Adoi:10.1016/j.jelechem.2008.07.030

* Corresponding author. Tel./fax: +86 25 8359 4862E-mail address: hychen@nju.edu.cn (H.-Y. Chen).

A simple one-step biomimetic coprecipitation method is reported to prepare a protein based biosensor byimmobilizing calcium phosphate/hemoglobin composite nanoparticles directly onto glassy carbon elec-trode surface. Atomic force microscopy (AFM), attenuated total reflection spectra (ATR) and electrochem-ical method were used to characterize the as-prepared composite nanoparticles. Hb in the composite filmkept their biological activity well, realized its direct electrochemistry with the formal potential of�0.363 V in pH 7.0 phosphate buffer solution and gave excellent electrocatalytic performance to thereduction of hydrogen peroxide, by which the mediator-free biosensors could be fabricated. Comparedto other immobilization methods, this system not only avoids the tedious synthesis of nanoparticlesand the use of reagent detrimental to biomolecules but also possesses the advantages of simple prepara-tion and low cost.

� 2008 Elsevier B.V. All rights reserved.

1. Introduction

Nanomaterials have attracted considerable interest in recentyears for their unique optical, electronic, chemical, and mechanicalproperties. Biomolecule-nanomaterial composite systems, combin-ing the recognition and catalytic properties of biomolecules withunique functions of nanomaterials, could be exploited for numer-ous applications in biomolecular electronics, biosensors, bioactua-tors, and biomedicine [1–3]. Recently, a large number of biosensorsbased on composite materials by combining the enzymes, antibod-ies and DNA with metallic or semiconductor nanoparticles havebeen developed [1–9]. However, at present, the most common pro-cedure used in the preparation of composite materials includes thepreparation of nanomaterials and the immobilization of biomole-cules on them through either physical adsorption or chemicalcross-linking. In many cases, such methods would employ toxic re-agents and require tedious procedures. Therefore, developing goodbiocompatible carriers and exploring new strategies for proteinimmobilization still are prevailing subjects in the design ofbiosensors.

As one of the most ideal biocompatible materials, Calcium phos-phate compounds (CaPs) were often used to immobilize proteins.Furthermore, due to the unique multiple-sites bindings to proteinsfor the abundant Ca2+ and the presence of PO3�

4 , CaPs can carrymore proteins and were used in biosensors [10–12]. In our previ-ous report, we constructed vertically oriented CaP–PDDA nano-

ll rights reserved.

.

walls on glassy carbon electrode (GCE) surfaces byelectrodeposition method and further immobilized hemoglobin(Hb) on them. The resulting Hb/CaP–PDDA film not only realizedthe direct electron transfer of Hb but also facilitated the enzymaticreaction toward many substances based on keeping the high cata-lytic performance of immobilized Hb [10].

In recent years, biomimetic coprecipitation as a simple and con-venient method for the formation of biomolecules-CaP compositeswithout any denaturation occurring has attracted much interest. Itis generally fulfilled by immersing substrates in a protein-contain-ing supersaturated calcium phosphate solution for a certain time.However, the researches mostly focused on the study of mecha-nism of biomimetic coprecipitation process and their biomedicalapplications [13–22]. In this paper, we report the one-step prepa-ration of CaP/Hb composite nanoparticles on the surface of GCE bybiomimetic coprecipitation, and their use for bioelectronic sensingapplications.

2. Experimental section

2.1. Regent

Human hemoglobin (Hb, MW 66,000) and hemin (MW 651.96)were purchased from Sigma and used without further purification.Hydroperoxide (H2O2) was freshly prepared before use. Phosphatebuffer solution (PBS, 25 mM) was prepared by mixing the solutionsof K2HPO4 and NaH2PO4. Different pH values were adjusted withH3PO4 or NaOH. Other chemicals were of analytical grade. All solu-tions were prepared with ultra-pure water.

80 H. Zhang et al. / Journal of Electroanalytical Chemistry 624 (2008) 79–83

2.2. Instruments

Atomic force microscopy (AFM) in the tapping mode was con-ducted with a Molecular Imaging PicoScan system (MolecularImaging Inc, Tempe, AZ) on GC plates. Ultraviolet and visible(UV–vis) absorption spectra were recorded with a Lambda 35UV–vis spectrometer (Perkin–Elmer instruments, USA). Fouriertransform infrared (FTIR) spectroscopic measurements were per-formed on a Bruker model VECTOR22 Fourier transform spectrom-eter, using KBr pressed disks. Attenuated total reflection (ATR)spectra were obtained on a NEXUS 670 (Nicolet).

Electrochemical measurements were performed on an AutolabPGSTAT-30 potentiostat/galvanostat (Eco Chemie BV, Utrecht, TheNetherlands), conventional three-electrode system was used witha modified glassy carbon electrode (GCE, d = 3 mm) as a workingelectrode, a platinum wire as an auxiliary electrode, and a satu-rated calomel electrode (SCE) as a reference electrode againstwhich all potentials were measured. Cyclic voltammetric measure-ments were done in an unstirred electrochemical cell at room tem-perature. All solutions were deoxygenated by bubbling highly purenitrogen for at least 20 min and a nitrogen atmosphere was keptduring the measurements.

2.3. Preparation of CaP/Hb modified GCE

The CaP/Hb modified GCE was prepared according to reference[14] with slight modification (Fig. 1). Briefly, The bare GCEs werepolished with 1.0, 0.3, and 0.05 lm alumina slurry followed byrinsing thoroughly with ultra-pure water and then allowed todry at room temperature. Cleaned GCE was immersed in HCP solu-tion for 12 h at 4 �C, and was rinsed with ultra-pure water, dried atroom temperature. The CP solution was prepared by dissolving

Fig. 1. Scheme of the CaP/Hb composite preparation by biomimetic coprecipitationmethod.

Fig. 2. AFM images of bare GC (a) and GC im

NaCl (142 mM), K2HPO4 � 3H2O (1.50 mM), and CaCl2 (3.75 mM)in ultra-pure water, buffering to pH 7.40 at 25 �C usingtris(hydroxymethyl)aminomethane (TRIS) (50 mM) and aqueous1 M HCl solution. The HCP solution was prepared by adding Hbto the CP solution to get 3 mg ml�1 Hb solution, which was storedat 4 �C.

3. Results and discussion

3.1. AFM characterization

Generally, there are two steps occurred in biomimetic coprecip-itation process in previous reports. Firstly, substrates were modi-fied with CaP precursors [13–20] or functional groups [21,22]which effectively induce CaP nucleation; secondly, these substrateswere immersed in CP solution containing proteins for a certaintime, as a result, a CaP/protein composite layer formed on thematerial surface. In this case, CaP/Hb composites formed on thesurface of GCE via one step coprecipitation without introducingany precursors or functional groups. AFM was used to monitorthe formation of the composite film on the GC substrate, As seenin Fig. 2a, The surface of GC is rough, less than 3 nm in heightand some carbon particles are observed from the polishing of theGC substrate. After 12 h immersion in the HCP solution, plenty ofsmall nanoparticles sized about 25 nm are superposed over theGC surface, with 4–5 nm in height (Fig. 2b), implying that CaP/Hbcomposites successfully formed on the GC substrate. In addition,the thickness of the film could be controlled by variation of theimmersion time. As shown in Fig. 3, the increase of the depositiontime from 30 min to 18 h resulted in the increase of the filmthickness.

3.2. Optical spectroscopy characterization

In order to get more information about the conformationalstructure of Hb, UV–vis spectra was employed. Fig. 4 shows thespectra of Hb in PBS solution (curve a) and HCP solution (curveb). When Hb was in pH 7.4 PBS or HCP solution, the Soret bandboth lied at 408 nm. For comparison, we tested the UV–vis spectraof hemin, a free heme group, the Soret band of which was observedat 388 nm in solution (curve c). Since the shape and position of theSoret absorption bands are sensitive to the variation of the micro-environment around the heme site [23]. No obvious blue shift ofSoret band of Hb in HCP solution toward the band of free heminsuggested that the heme prosthetic group of Hb does not splitout from Hb polypeptide in HCP solution.

mersed in the HCP solution for 12 h (b).

Fig. 3. AFM images of bare GC (a) and GC immersed in the HCP solution for 0.5 (b), 6 (c), 12 (d), and 18 h (e).

300 400 500 600

Rel

ativ

e A

bsor

banc

e

Wavelength (nm)

cb

a

Fig. 4. UV–vis spectra of Hb in pH 7.4 PBS (a), HCP solution (b) and hemin in pH 9.0PBS (c).

800 1000 1200 1400 1600 1800 2000

Tra

nsm

ittan

ce

Wavelength (cm-1)

p-o

amide I amide II

a

b

Fig. 5. ATR spectra of Hb/CaP film (a) and FTIR spectra of Hb (b).

H. Zhang et al. / Journal of Electroanalytical Chemistry 624 (2008) 79–83 81

Fig. 5 gives ATR spectra of CaP/Hb nanoparticles and the FTIRspectra of native Hb. The peak at 1033.7 cm�1 in Fig. 5 indicatesthe existence of phosphate groups [13], the adsorption bands foramide I and amide II of Hb in the CaP/Hb nanoparticles are locatedat 1648.1 cm�1 and 1541.4 cm�1 (Fig. 5a), which confirm the for-mation of Hb/CaP nanocomposite. It is known that the shape andposition of infrared bands of amide I (1600–1700 cm�1) and amideII (1500–1600 cm�1) can provide detailed information on the sec-

ondary structure of polypeptide chain of proteins [24]. Theseabsorption bands are very similar to those of the native protein(1652.7 and 1539.5 cm�1) (Fig. 5b), indicating an actually undis-turbed second structure of the protein bound in the CaP/Hbnanoparticles.

For investigating the formation mechanism of CaP/Hb, we im-mersed GCE in the CP solution without proteins. The results ofAFM and the ATR both showed that no CaP layer formed on GCE

-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4

-3

-2

-1

0

1

2

3

4

5

1.2 1.6 2.0 2.4 2.8 3.2

-0.8

-0.4

0.0

0.4

logi

pc (μ

A)

logν (mV.S-1)

a

i (μA

)

E (V)

I

Fig. 7. CVs of Hb/CaP (a) modified GCE at various scan rates in pH 7.0 PBS. 20, 40,60, 80, 100, 300, 500, 800, 1000 mV s�1 (a)–(i). Inset: The linear relationship oflogarithm of cathodic peak currents (Ipc) vs. logarithm of scan rate (m).

82 H. Zhang et al. / Journal of Electroanalytical Chemistry 624 (2008) 79–83

without Hb. It indicated that the proteins do play an important rolein the formation of CaP/Hb. Just as Oyane [14] have reported thatlaminin–apatite nanocomposite was prepared in a laminin-con-taining CP solution through the chemical bonding between gelatinand calcium ions of apatite, it considered that a similar interactionoccurs between Hb and CaP. As the GCE was immersed in Hb-con-taining CP solution, some proteins were adsorbed on the surface ofthe GCE [25], the functional groups of Hb were thought to induceheterogeneous CaP nucleation. Once the nuclei formed, they grewspontaneously by consuming the calcium and phosphate ions inthe HCP solutions[13–22], Meanwhile, Hb was stabilized and dis-tributed in the composite layer through the chemical bonding withthe surrounding CaP crystals. As a result, Hb/CaP composites wereformed on the GCE.

3.3. Direct electrochemistry of Hb

The direct electron transfer of Hb in nanocomposite was inves-tigated. In pH 7.0 PBS, bare GCE (curve a in Fig. 6) or GCE afterimmersion in CP solution for 12 h (curve b in Fig. 6) showed no re-dox response, however, CaP/Hb modified GCE displayed a pair ofwell-defined redox peaks at about �0.428 V and �0.298 V (curvec in Fig. 6), which are in accordance with the characteristic ofFeIII/FeII redox couples of heme proteins. The shapes of the reduc-tion and oxidation peaks were nearly symmetric. The E00 , as theaverage of oxidation and reduction peak potentials, was�0.363 V. The amount of redox Hb in the nanocomposite wasgreatly depended on the immersion time. The peak currents ofHb increased with the increase of immersion time from 6 h to12 h, and reached plateau when the immersion time was over12 h. Here an optimal immersion time of 12 h was selected forpreparation.

3.4. Electrochemical and electrocatalytic behaviors of Hb in CaP/Hbfilm

With the increase of scan rate (t) from 20 to 1000 mV s�1, thepeak potentials for Hb did not change and the redox peak currentsincreased (Fig. 7). The inset of Fig. 7 showed a linear relationshipbetween log Ipc and log m with a slope of 0.67, which is more thanthe slope of 0.5 for ideal diffusion-controlled reaction and less thanthe slope of 1 for ideal thin layer electrochemistry [26]. Thus theredox reaction rate is controlled by both the diffusion processand the surface electron transfer process [27]. The peak current

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4-0.6

-0.4

-0.2

0.0

0.2

0.4

i (μA

)

E (V)

ab

c

Fig. 6. CVs of bare GCE (a), GCE in PBS solution after immersion in CP (b) and HCP(c) solution for 12 h.

was linearly proportional to the scan rate in the range of 100–1000 mV s�1. According to Laviron equation [28]

Ip ¼ n2F2mCA=4RT ð1Þ

From the slope of the Ip–m curve, the average surface coverage (C) ofelectro-active Hb was estimated to be 2.06 � 10�11 M cm�2, whichis close to the monolayer coverage (the theoretical monolayer cov-erage of 1.89 � 10�11 M cm�2) [29]. In addition, according to theLaviron equation [28]

Ks ¼ mnFm=RT ð2Þ

The surface electron transfer rate constant Ks can be calculated,where m is a parameter related to DEp. With this formula, the sur-face electron transfer rate constant of 0.71 s�1 was obtained forCaP/Hb, smaller than that of Hb immobilized on mesoporouscarbon/whisker-like carbon (MCWC) composite of 2.07 s�1 [30],but larger than that of Hb immobilized on carbon nanotube of0.49 s�1 [31].

The pH of PBS solution played an important role in the electro-chemical behavior of Hb. The increase of pH led to a negative shiftof the potential of both reduction and oxidation peaks (Fig. 8). The

-1.6 -1.2 -0.8 -0.4 0.0 0.4

-0.8

-0.4

0.0

0.4

0.8

3 4 5 6 7 8 9-0.5

-0.4

-0.3

-0.2

Eo'

( V

)

pH

pH9.0 pH3.0

i (μA

)

E (V)

Fig. 8. CVs of Hb/CaP modified GCE at various pH values. Inset: Plot of pH values vs.E00 .

0 500 1000 1500 2000 2500-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0 2 4 6 8 10

0.00.10.20.30.40.50.6

i-1 (

mA

-1)

[H2O2]-1 (M-1)

i (μA

)

t (s)

2.0mM

1.0mM

0.5mM10μM

4.0μM

2.0μM

1.0μM0.4μM

0.1μM 0.2μM

Fig. 9. A typical amperometric i–t curve of CaP/Hb modified GCE at �250 mV in2.0 ml 25 mM PBS solution with pH 7.0. Inset: The linearity relationship betweenthe reciprocals of current and H2O2.

H. Zhang et al. / Journal of Electroanalytical Chemistry 624 (2008) 79–83 83

E00 has a linear relationship at pH range of 3.0 to 9.0 with a slope of–50.0 mV pH�1. This value was close to the theoretical value of�58.0 mV pH�1 for a reversible proton-coupled single electrontransfer [32]. This showed that such an electrode process is a singleelectron transfer accompanying a single proton between the elec-trode and the heme Fe (III) of Hb. In addition, the CV peak currentsof Hb/CaP was almost unchanged after investigating the CV behav-ior of the modified electrode in low pH value PBS (pH 6,5,4,3) andthen returning to the original pH 7.0 PBS, which reflects an excel-lent stability of the composite film in acidic solution.

In order to survey the activity of Hb immobilized in CaP/Hbcomposite nanoparticles, we choose H2O2 as an example to exam-ine the biocatalytic ability of Hb. When H2O2 was added to a pH 7.0buffer solution, an increase in the reduction peak was observedwith the decrease of the oxidation peak for Hb. This result showsthat the CaP/Hb modified GCE could effectively catalyze the reduc-tion of H2O2. The amperometric response of CaP/Hb modified GCEwith successive additions of H2O2 to 25 mM, pH 7.0 PBS at an ap-plied potential of �250 mV was shown in Fig. 9. Upon the additionof H2O2, the reduction current increased steeply to reach a stablevalue. The modified GCE achieved 95% of the maximum steady-state current within 10 s, which was attributed to the fast diffusionof the substrate in the CaP film. A linear relationship between [i]�1

and [H2O2]�1 is shown in the inset of Fig. 9, The corresponding cal-ibration plot is linear over 0.1 lM to 2.6 mM with a correlationcoefficient of 0.9994 and detection limit of 0.1 lM was obtainedat a signal-to-noise ratio of 3 (Fig. 9). The apparent Michaelis–Men-ten constant ðKapp

M Þ provides an indication of the enzyme–substratekinetics and a way to compare one H2O2 sensor with others. It canbe calculated from the Lineweaver–Burk equation [33]

1=Iss ¼ I=Imax þ KappM =ImaxC ð3Þ

where Iss is the steady-state current after the addition of substrate,C is the bulk concentration of the substrate, and Imax is the maxi-mum current measured under saturated substrate conditions. Kapp

M

can be obtained by the analysis of slope and intercept of the plotof the reciprocals of the steady-state current versus H2O2 concen-tration. The Kapp

M for Hb/CaP modified GCE was found to be8.08 lM, which was smaller than that of Hb–MCWC/GC electrode

[30]. It should also be pointed out that CaP/Hb modified GCE dis-plays a good stability with a relative standard deviation of 4.6%for eight independent determinations for 50 lM H2O2 solution.

4. Conclusion

In summary, CaP/Hb composite nanoparticles has been fabri-cated on the surfaces of GCEs via a biomimetic coprecipitationmethod. This method is simple and convenient. The resulted CaP/Hb composite nanoparticles not only realized the direct electrontransfer of Hb but also retained its bioactivity. Such an architec-tural approach and formed composite nanoparticles have found agood application in the biosensing fields.

Acknowledgements

Financial support from the National Natural Science Foundation(20675037, 20435010, 20635002, 20775033), the 973 Program(2007CB936404, 2006CB933201), the National Natural ScienceFunds for Creative Research Groups (20521503) and the programfor New Century Excellent Talents in University (NCET) of Chinaare gratefully acknowledged.

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