l -Cysteine composite film

Colloids and Surfaces B: Biointerfaces 86 (2011) 339–344

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Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6 /l-Cysteine composite film Lingsong Lu, Bei Liu, Shenfeng Li, Wei Zhang, Guoming Xie ∗ Key Laboratory of Medical Diagnostics of Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China

a r t i c l e

i n f o

Article history: Received 13 December 2010 Received in revised form 27 March 2011 Accepted 8 April 2011 Available online 17 April 2011 Keywords: Myeloperoxidase l-Cysteine Ionic liquids CeO2 Immunosensor

a b s t r a c t An electrochemical immunosensing assay for myeloperoxidase (MPO) determination in human serum has been developed. Firstly, l-Cysteine was initially electropolymerized on an Au electrode to form l-Cysteine film. After that cerium dioxide (CeO2 ) dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6 ) were immobilized on the l-Cysteine film. Then the negatively charged nanogold particles were adsorbed onto the membrane via the positive charge of CeO2 , which aimed at assembling more antibody of MPO (anti-MPO). The resulting immunosensor showed a high sensitivity, broad linear response to the MPO concentration comprised between 10 ng/mL and 400 ng/mL with a detection limit of 0.06 ng/mL. Moreover, the surface morphology of the electrode was studied by means of a scanning electron microscope and the electrochemical properties of the fabricated immunosensor were further characterized by cyclic voltammetry. Also, factors influencing the performance of the resulting immunosensors were studied in detail. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Acute coronary syndrome (ACS) involves ST elevation myocardial infarction, non ST elevation myocardial infarction and unstable angina, which provides a threat to human health. Early ACS diagnosis is a crucial step for effective controlling and treatment of the disease. Conventionally, Myoglobin (Myo), Troponin I (cTnI) and Creatine Kinase-MB (CK-MB) in human serum were detected to distinguish vulnerable atheroma in clinical diagnosis. But they all increased after myocardial infarction occurred. Myeloperoxidase (MPO), a heme peroxidase has been given much attention as a prognostic indicator of suspected ACS [1,2]. In patients with ACS, increased serum levels of MPO powerfully predict an increased risk for subsequent cardiovascular events even in the absence of established indicators of myocardial necrosis [3,4]. Therefore, the development of highly sensitive MPO assay has great potential in earlier detection of acute myocardial infarction and in risk prediction in patients with ACS. The conventional methods used for MPO quantification include radio-immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Despite exhibiting high sensitivity and selectivity, these traditional immunoassays still have some drawbacks, such as time consuming, the dependence of sophisticated and expensive equipment, and the demand for skilled professionals

∗ Corresponding author. Tel.: +86 23 68485388; fax: +86 23 68485005. E-mail address: [email protected] (G. Xie). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.04.017

[5]. Electrochemical immunosensors combined the specificity of immunoreaction with the sensitivity of electrochemical method, which have been considered suitable for the determination of MPO. Compared with conventional immunoassay methods, electrochemical immunosensor exhibited several advantages, such as high sensitivity, fast analysis, small analyte volume and simple pretreatment [6–8]. With the recent development in nanotechnology, nanoparticles have been widely used in the construction of immunosensors [9]. Gold nanoparticles have received much attention because of its unique chemical and physical properties such as large specific surface area, good biocompatibility and high surface free energy [10,11]. Nanogold cannot only firmly adsorb antibody or enzyme, but also retain their biological activity [12,13]. Recently, cerium dioxide (CeO2 ) has attracted considerable interest and has been successfully employed in enzyme [14] or antibody [15] immobilization due to the high isoelectric point (IEP ∼ 9.2), which can be helpful to immobilize desired antibodies of low IEP via electrostatic interactions [16]. l-Cysteine is an amino acid that plays an important role in biological systems. Liao et al. used l-Cysteine as an electron transfer promoter to compose a self-assembled monolayer (SAM) [17]. However, the SAM is unstable, and low-molecular-weight l-Cysteine may leach out from the electrode surface, influencing the performance of the immunosensor [18,19]. RTILs, as green solvents, have been gaining increasing attention in electroanalytical chemistry because of their negligible vapor pressure, outstanding chemical and thermal stability, high conductivity, and low toxicity

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[20–22]. BMIMPF6 is a kind of hydrophobic RTIL. After BMIMPF6 was coated on the electrode surface, it could form a hydrophobic membrane to avoid the direct contact with the buffer solution. Then l-Cysteine modified on the electrode cannot leach out from the electrode surface. A composite film consisting of multi-walled carbon nanotubes and ionic liquid modified on gold carbon electrode is prepared for the fast detection of glucose [23]. Xiang et al. studied the direct electron transfer of cytochrome c in ionic liquid/carbon nanotubes nanohybrid film [24]. Some electroanalytical chemistry methods have been reported to detect MPO. Windmiller et al. employed 3,3 ,5,5 tetramethylbenzidine (TMB) as a redox mediator for the determination of MPO in the presence of a peroxide substrate [25]. Lin et al. proposed a biosensor assay system using diatoms as key component to selectively and quantitatively detect C-reactive protein (CRP) and MPO [26]. However, diatoms cells were cultured in f/2 seawater medium at 20 ◦ C under continuous photoperiod, which needed entail complex, labor-intensive preparation procedure. In our prior work, we fabricated an immunosensor based on N,N-dimethylformamide (DMF), multi-wall carbon nanotubes (MWCNTs), CeO2 and 1-ethyl-3-methyl imidazolium tetrafluoroborate (EMIMBF4 ) composite film for the determination of MPO [27]. This method has several advantages such as a wide linear detection range and no need for multiple labeling procedure. Despite these advantages, the immunosensor still has some limitations: Firstly, the double layer composite film is too thick, which is not good for efficiently improving the electron transfer between the composite film and the electrode surface; Secondly, EMIMBF4 is a kind of hydrophil RTIL, which is apt to dissolve into the buffer solution or human serum sample. These limitations can significantly impact the sensitivity of the immunosensor. In the present work, BMIMPF6 was coated on the electrode surface to form a hydrophobic membrane, which can help prevent the leakage of the other modification materials from the electrode surface. After the nanogold and nanoCeO2 particles were immobilized on the electrode surface, they help adsorb more anti-MPO, aiming at improving the sensitivity of the immunosensor. With the aim to explore the excellent film-forming ability and the good conductivity of BMIMPF6 and the affinity of nanoCeO2 toward the anti-MPO, we first used nanogold/nanoCeO2 BMIMPF6 /l-Cysteine composite film as immobilization matrix to fabricate an electrochemical immunosensor, finding that the immunosensor exhibited a wonderful electrochemical response to trace the amount of MPO. In the present work, the immunosensor employs the current changes before and after the immnoreaction to quantitatively detect MPO. This immunoassay protocol possesses several attractive improvement, such as relative higher sensitivity, a wider linear detection range and lower detection limit for MPO. This is because the composite film offers a larger surface area, allowing an increased immobilization amount of anti-MPO on the electrode surface. Compared with the previous work, we simplified the electrode modification procedure and reduced electrode modification materials. Thus, fabricating such an immunosensor, the electrode modification procedure is fewer and the cost is lower. Moreover, the factors influencing the performance of the obtained immunosensor were also discussed in detail.

2. Experimental 2.1. Reagents and materials NanoCeO2 (spherical, particle diameter: 20 nm) was obtained from Beijing Nachen S&T Co. (Beijing, China). Chloroauric acid (HAuCl4 ), sodium citrate, MPO, anti-MPO and Bovine serum albumin (BSA, 96–99%) were obtained from Sigma (St. Louis, MO, USA).

BMIMPF6 was purchased from Lanzhou Greenchem ILS, LICP. CAS. China (Lanzhou, China). l-Cysteine and acetone were purchased from Shanghai Reagent Company (Shanghai, China). Double distilled water was used throughout this study. Phosphate-buffered solutions (PBS) at various pH values (4.5–7.5) were prepared using 0.1 M NaH2 PO4 , 0.1 M Na2 HPO4 and 0.1 M KCl. The standard MPO was diluted into different concentrations by PBS. Serum specimens were gifted by the First Affiliated Hospital, Chongqing Medical University. The blood samples were centrifuged for 10 min at room temperature at 1000 × g. The serum was kept frozen in small aliquots at −20 ◦ C. Immediately before performing the immunosensor assay, samples were thawed. All reagents were of analytical grade unless otherwise stated. 2.2. Apparatus and measurements A ␮AUTOLAB ␤ Electrochemistry Workstation (Eco Chemie, Netherlands) was used for cyclic voltammetric measurements. A three-electrode system contained a modified Au electrode (3 mm in diameter) as the working electrode, platinum (Pt) wire as the auxiliary electrode and Ag/AgCl as the reference electrode. The electrochemical characterizations and measurements on the modified electrode were carried out by using cyclic voltammetry from −0.2 to 0.8 V (vs. Ag/AgCl) in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− . Scanning electron microscopy (SEM) was carried out using a Hitachi S-4800 Scanning Electron Microscope (Hitachi, Japan). 2.3. Preparation of gold nanoparticles The gold nanoparticles were prepared according to the literature [28]. 1 mL of 1.0 wt.% HAuCl4 solution was initially added to 99 mL water and then heated until boiling. 2.5 mL of 1.0 wt.% sodium citrate solution was injected into the boiling solution quickly. The mixture was heated for 15 min continuously until the solution color became claret. The obtained gold colloids were cooled at room temperature, and stored at 4 ◦ C when not in use. 2.4. Preparation of nanoCeO2 -BMIMPF6 nanocomposites Briefly, appropriate amount of nano-sized CeO2 (final concentration 1.0 mg/mL) was dispersed in BMIMPF6 solution (BMIMPF6 was diluted by acetone, Vacetone :VBMIMPF6 = 1:3). After sonicated for 2 h, the well-dispersed nanoCeO2 -BMIMPF6 nanocomposites were obtained. The resulted nanoCeO2 -BMIMPF6 nanocomposites were kept at 4 ◦ C. 2.5. Fabrication of the immunosensor The Au electrode was successively polished to obtain a mirrorlike surface with 0.3 ␮m and 0.05 ␮m alumina slurry. Following that, the electrode was rinsed by double distilled water. Then, the electrode was sonicated in double distilled water, ethanol and double distilled water for 5 min, respectively. The electrode was immersed into a solution of 10 mM l-Cysteine. 30 voltammetric cycles were carried out between −0.2 and 1.5 V at a scan rate of 100 mV/s. After that, 6 ␮L of nanoCeO2 -BMIMPF6 nanocomposites was coated onto the surface of the Au electrode modified by l-Cysteine. After the electrode was dried at room temperature, the nanoCeO2 -BMIMPF6 /l-Cysteine/Au was immersed into gold colloids at 4 ◦ C for 2 h. The gold nanoparticles were immobilized on the electrode by the positive charge of CeO2 . Following that, the nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine nanocomposites film modified Au electrode was dipped into the anti-MPO solution (1 mg/mL) at 4 ◦ C for 12 h. Finally, the modified electrode was incubated into 2.5 wt.% BSA–PBS (pH 7.0) solution for 60 min at

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Scheme 1. Schematic drawing of the stepwise immunosensor fabrication process: (a) electropolymerization of L-Cysteine; (b) dropping of nanoCeO2-BMIMPF6 nanocomposites; (c) adsorption of nanogold; (d) anti-MPO loading; (e) BSA blocking.

room temperature to block the remaining active sites of nanogold and nanoCeO2 . The as-prepared immunosensor was stored at 4 ◦ C when not in use. The construction procedure of the immunosensor is schematically illustrated in Scheme 1.

d c e f b a

200 150 100

3.1. Morphology characterization of the different modified electrodes The morphologies of nanogold/nanoCeO2 -BMIMPF6 /lCysteine/Au and anti-MPO/nanogold/nanoCeO2 -BMIMPF6 /lCysteine/Au were studied by means of SEM. Fig. 1A shows that a relatively homogeneous nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine composite film was distributed over the surface of the Au electrode, which was attributed to the good solubility and dispersing ability of BMIMPF6 toward nanogold and nanoCeO2. It can be seen from Fig. 1A that nanoCeO2 are brighter than nanogold with particle diameter of 20 nm. The modification of the electrode with double nanoparticles greatly increased the amount of the anti-MPO immobilization. After coated with anti-MPO, the Au electrode surface became more uniform and dim (Fig. 1B). It explained that anti-MPO had been adsorbed on the electrode surface by nanogold and nanoCeO2 .

Current / μA

3. Results and discussion

50 0 -50 -100 -150 -200 -250 -0.2

0.0

0.2

0.4

0.6

0.8

Potential / V Fig. 2. Cyclic voltammograms of the stepwise immunosensor fabrication process in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− : (a) Au electrode; (b) l-Cysteine/Au; (c) nanoCeO2 (d) nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine/Au; BMIMPF6 /l-Cysteine/Au; (f) BSA/anti(e) anti-MPO/nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine/Au; MPO/nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine/Au. Scan rate: 50 mV/s. All potentials are given vs. Ag/AgCl.

3.2. Electrochemical characterization of the immunosensor Fig. 2 shows the cyclic voltammetry electrochemical performances of different modified electrodes. It can be seen from the curve a that a pair of well-defined redox peaks appeared when the Au electrode was immersed in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− as the redox probe. As can be

seen from curve b, the current increased after l-Cysteine was electropolymerized on the surface of the Au electrode. This suggested that l-Cysteine promoted electron transfer and enhanced the conductivity of the electrode. Compared with curve b, the current of curve c increased after nanoCeO2 -BMIMPF6 composite film was coated on the surface of the Au electrode, which was attributed to

Fig. 1. The SEM images of (A) Au electrode covered with nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine composite film, (B) same setup as (A) following immobilization of anti-MPO.

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Current change / μA

A

12 10 8 6 4 2 0 4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

that BMIMPF6 can greatly improved the conductivity of the composite film. When the electrode was dipped into the gold colloids, the current further increased (curve d). After the anti-MPO was immobilized onto the electrode surface, the redox peaks decreased as the immune-protein film acted as an inert electron and mass transfer blocking layer (curve e). At last, a further decrease can be observed from curve f after BSA was used to block the possible remaining active sites. Furthermore, cyclic voltammograms of the prepared immunosensor in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− at different scan rates were observed in Fig. 3. It can be seen that pairs of roughly symmetric anodic and cathodic peaks appeared with almost equal peak currents in the scan rate range from 20 to 200 mV/s. The peak-to-peak separation also increased with the increment of the scan rate. Linear relationships with good correlation coefficients were observed between the peak currents and the square root of scan rates, which is shown in the inset of Fig. 3. The oxidation and reduction peak currents increased linearly with the linear regression equations as Ipa = 55 + 27 v1/2 (mV/s)1/2 (n = 7, r = 0.9997), Ipc = −55 − 29 v1/2 (mV/s)1/2 (n = 7, r = −0.9998), respectively, suggesting that the reaction was a quasi-reversible diffusion-controlled process. 3.3. Optimization of experimental conditions To select the optimum concentration of anti-MPO solution, the standard anti-MPO was diluted into different concentrations. As concentration of anti-MPO solution increased, more anti-MPO can be immobilized on the electrode surface. After the concentration reached 1 mg/mL, the amount of anti-MPO immobilized on the electrode surface did not increase anymore. So we selected 1 mg/mL as the optimum concentration of anti-MPO solution. The effect of pH value of the supporting electrolyte on the current responses of the fabricated immunosensors was studied. As shown in Fig. 4A, the current change increased with the increment of pH value from 4.5 to 7.0 and then decreased. This was because anti-MPO is a kind of protein and it only can keep its bioactivity at biocompatible pH. Highly acidic or alkaline surroundings would damage the bioactivity of the immobilized antibody. When the pH of the buffer solution was adjusted to 7, anti-MPO immobilized on the electrode surface could adsorb more MPO. The amount of the formative anti-MPO/MPO immunocomplex achieved the max-

B 12

Current change / μA

Fig. 3. Cyclic voltammograms of the developed immunosensor at different scan rates in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− . From inner to outer: 20, 40, 60, 80, 100, 150, 200 mV/s. Inset: the dependence of redox peak currents on the square root of scan rates.

10 8 6 4 2 20

25

30

35

40

T /ºC Fig. 4. Influence of the pH of buffer solutions (A) and the incubation temperature (B) on the responses of the immunosensor to 100 ng/mL MPO in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− at 50 mV/s.

imum value. The diffusion of ferricyanide toward the electrode surface was enormously hindered, which caused the most obvious current change. So we set the optimum pH at 7.0. The influence of the antigen-antibody incubation temperature on the amperometric response was also studied. When the temperature achieved 37 ◦ C, we got the most obvious current change in Fig. 4B. So the optimal temperature was 37 ◦ C. 3.4. Amperometric response and calibration curve When the immunosensor was incubated in the MPO solution for 10 min, a dramatic decrease in current is observed (Figure 5). This was attributed to the formation of anti-MPO/MPO immunocomplex, which acted as the inert electron and mass transfer blocking layer and hindered the diffusion of ferricyanide toward the electrode surface. The detection principle was based on the change of oxidation peak current response (Ipa ) before and after the antibody-antigen reaction, which was evaluated as the following equation:Ipa = I0 − In , where I0 was the response current before the immunoreaction and In was the response current after the immunoreaction (n = 300 ng/mL). As shown in Fig. 6, the current changes before and after the immunoreaction were proportional to the concentrations of MPO in the linear range of 10–400 ng/mL and the linear regression equation is Ipa = 6.3 + 0.026 CMPO with a detection limit of 0.06 ng/mL

L. Lu et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 339–344

a b

Current / μA

100

Δ Ipa=I0- In

I0 In

c

Current change / μA

150

343

50 0 -50 -100 -150

15

10

5

-200 -0.2

0.0

0.2

0.4

0.6

0.8

0

Potential / V

a

Fig. 5. Cyclic voltammograms of the different modified Au electrodes in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− : (b) BSA/anti(a) anti-MPO/nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine/Au, MPO/nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine/Au and (c) (b) incubed with 300 ng/mL MPO. Scan rate is 50 mV/s.

b

c

d

e

f

g

Fig. 7. Comparison of the peak current changes by detecting 100 ng/mL MPO (a) containing the same concentration of Myo (b), cTnI (c), CK-MB (d), CRP (e), HCG (f) and l-Cysteine (g).

at a signal to noise ratio of 3 (where  is the standard deviation of a blank solution, n = 10, r = 0.9967).

changes in the MPO solution with and without interference showed a difference of less than 5%, which obviously indicated that the selectivity of the MPO immunosensor based on the highly specific antigen-antibody immunoreaction was satisfactory.

3.5. Reproducibility and selectivity

3.6. Regeneration and stability of the immunosensor

The repeatability and reproducibility of the proposed immunoassay were evaluated by the variation coefficients (CVs) of intra- and inter-assays. The intra-assay precision of the proposed immunosensor was evaluated by successively analyzing three MPO concentration levels 5 times per run. The CVs of intra-assay with this method were 2.8%, 1.9% and 3.8% at 50, 100 and 200 ng/mL of MPO, respectively. Similarly, the inter-assay CVs on five immunosensors used independently were 3.1%, 4.4% and 4.1% at 50, 100 and 200 ng/mL of MPO, respectively. These results suggested that the immunosensor showed acceptable precision and fabrication reproducibility. Selectivity is an important property of the immunosensor. In order to investigate the selectivity, the immunosensor was respectively incubated with a solution of 100 ng/mL MPO containing one of the following: Myo (100 ng/mL), cTnI (100 ng/mL), CK-MB (100 ng/mL), CRP (100 ng/mL), HCG (100 ng/mL) and l-Cysteine (100 ng/mL). The results are shown in Fig. 7. The peak current

In this study, after detecting MPO, the immunosensor was immersed in a stirring 8 M urea for 5 min, and then washed with double distilled water to dissociate the antigen–antibody complex. After that the immunosensor was used to detect the same concentration of MPO. The immunosensor kept 90% of the original current after regenerated five times and the relative standard deviation (RSD) was 3.6%. The stability of the immunosensor was also investigated. When the immunosensor was stored in the refrigerator at 4 ◦ C for 10 days, the response still retained 92% of the initial. The good stability may contribute to the good biocompatibility of the nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine film. Moreover, the nanostructure of nanoCeO2 and nanogold greatly enhance the active surface available for anti-MPO binding over the geometrical area.

In order to investigate the ability of the immunosensor to detect MPO accurately, the recovery test was carried out. The samples were prepared using standard MPO solution. The developed immunosensor was respectively incubated with five different concentrations of MPO in the working buffer for 10 min. The results are shown in Table 1. As can be seen, the recovery was in the range of 96.0–104.5%, which indicated that the immunosensor was applicable for MPO detection in the working buffer. To investigate the possibility of the immunosensor used for practical analysis, 6 serum samples were detected by the developed immunosensor and the standard ELISA method. The paired t-test was used to evaluate these two immunoassay methods by com-

18 16

Current change / μA

3.7. Application to real sample analysis

14 12 10 8

Table 1 The recovery of the prepared immunosensor.

6 0

100

200

300

400

CMPO (ng/mL) Fig. 6. Calibration curve of the developed immunosensor toward different concentrations of MPO in the range of 10–400 ng/mL in 0.1 M PBS (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)6 3− /Fe(CN)6 4− at 50 mV/s.

Sample 1 2 3 4 5

Added (ng/mL) 5.0 10.0 20.0 40.0 80.0

Found (ng/mL) (mean ± RSD, n = 3) 4.8 10.45 19.42 39.32 80.65

± ± ± ± ±

3.5 2.8 4.3 3.7 4.5

Recovery (%) 96.0 104.5 97.1 98.3 100.8

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Table 2 MPO concentrations in serum samples tested by different immunoassays. Sample number

1 2 3 4 5 6

Proposed immunosensor (ng/mL)

ELISA (ng/mL)

11.3 30.4 80.5 123.7 150.3 232.2

10.75 29.9 81.4 116.8 155.2 236.5

Relative error (%) 5.1% 1.7% -1.1% 6.0% 3.2% 1.9%

paring the test results. Statistical analysis of these data is shown in Table 2 at the 95% confidence level. The relative deviations between the two methods were in the range of −1.1% to 6.0%, which indicated that the developed immunoassay might provide a feasible alternative tool for the determination of MPO in human serum in clinical analysis. 4. Conclusions In this present work, we fabricated a label-free amperometric immunosensor for the detection of MPO based on the immobilization of anti-MPO on an Au electrode modified by nanogold/nanoCeO2 -BMIMPF6 /l-Cysteine composite membrane, which greatly improved the electrochemical behavior and enhanced the sensitivity of the immunosensor. The presence of nanogold and nanoCeO2 particles provided a friendly microenvironment for the immobilization of biomolecules. This method has several advantages such as high sensitivity, a wide linear detection range for the detection of MPO and no need for multiple labeling procedure. Importantly, because this approach does not require sophisticated fabrication procedure, it is well suited for pointof-care testing and self-monitoring in both clinical and scientific research areas. Acknowledgements This work was financially supported by the Natural Science Foundation of Chongqing, China (CSTC, 2010BB5356) and Founda-

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