An electrochemical enzyme bioaffinity electrode based on biotin–streptavidin conjunction and bienzyme substrate recycling for amplification

Analytical Biochemistry 405 (2010) 121–126

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An electrochemical enzyme bioaffinity electrode based on biotin–streptavidin conjunction and bienzyme substrate recycling for amplification Yali Yuan, Ruo Yuan *, Yaqin Chai, Ying Zhuo, Lijuan Bai, Yuhong Liao Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China

a r t i c l e

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Article history: Received 20 April 2010 Received in revised form 19 May 2010 Accepted 21 May 2010 Available online 25 May 2010 Keywords: Signal amplification Multienzymatic catalysis Sensitivity Biotin–streptavidin Electrochemical immunosensor

a b s t r a c t A signal amplificatory electrochemical immunoassay with biotin–streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin–streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 1014 to 105 g ml1 was acquired with a relatively low detection limit of 3.5  105 g ml1 for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here. Ó 2010 Elsevier Inc. All rights reserved.

Sensitive quantitative detection of protein biomarkers is critical to many areas of biomedical research and diagnostics, systems biology, and proteomics. Various methods of signal amplification have been developed to enhance the signal so as to obtain highly sensitive biosensors, including nanocompounds [1–4], quantum dots [5,6], radioisotopes [7], fluorescence compounds [8], and enzymes [9–11]. Among them, enzyme labeling is the most commonly employed for signal amplification [12–14] because it not only performs transduction of biomolecular recognition event into an electrochemical signal but also allows its amplification according to the intrinsic catalytic activity of the enzyme. A series of enzyme labels, such as alkaline phosphatase (AP)1 [15–20], esterase [21], glucose oxidase (GOD) [22], horseradish peroxidase (HRP) [23–25], bilirubin oxidase [26], and diaphorase (DI) [27], have been

* Corresponding author. Fax: +86 23 68252277. E-mail address: [email protected] (R. Yuan). 1 Abbreviations used: AP, alkaline phosphatase; GOD, glucose oxidase; HRP, horseradish peroxidase; DI, diaphorase; PAPP, p-aminophenylphosphate; PAP, paminophenol; PQI, p-quinoneimine; NADH, nicotinamide adenine dinucleotide; bioIgG, biotin-labeled secondary antibody; bio-AP, biotinylated alkaline phosphatase; GA, glutaraldehyde; nano-TiO2, titania nanoparticles; APTES, 3-aminopropyltrimethoxysilane; BSA, bovine serum albumin; Tris, tris-hydroxymethylaminomethane hydrochloride; PBS, sodium phosphate buffer; CV, cyclic voltammetry; SCE, saturated calomel electrode; NH2–TiO2, amino-functional nano-TiO2; AFP, a-fetoprotein; HCG, human chorionic gonadotropin. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.05.025

successfully designed for signal amplification with low detection limits. Recently, researchers advanced a fascinating method with multienzymatic catalysis to improve the sensitivity of biosensors. The approach consisting in coimmobilization of two enzymes on the electrode surface could obtain a high amplification factor of more than 3000 times, which has been demonstrated by Limoges’s group [28]. In our previous work, bienzyme-functionalized threelayer composite magnetic nanoparticles for electrochemical immunosensors with HRP and GOD were designed for signal amplification [29], which obtained a high sensitivity and a low detection limit (reached to pg ml1). Although possessing the above advantages of the coimmobilization of bienzymatic system, coimmobilization of two enzymes on the electrode surface made the experimental procedure complicated. In the current study, a more practicable approach that consisted of two enzymatic amplifiers in an electrolytic cell was employed. In the electrolytic cell, the immobilized AP at immunosensor could hydrolyze electroinactive p-aminophenylphosphate (PAPP) substrate into the electroactive p-aminophenol (PAP). Thus, PAP was then oxidized at the electrode into p-quinoneimine (PQI) according to a (2e + 2H+) reaction. With the help of the enzymatic amplifier DI, PQI was reduced back to PAP, leading to a reversible cycle of PAP. Next, the oxidized form of DI was regenerated into its reduced native state by another enzymatic amplifier, nicotinamide adenine dinucleotide (NADH).

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Finally, a bienzymatic substrate recycling system was successfully constructed for signal amplification. In addition, to improve the amount of immobilized AP and further improve the sensitivity of the proposed immunosensor, the streptavidin–biotin system, a general paradigm advantageous for amplifying electrochemical signals was also adopted in this work. Biotin-labeled secondary antibody (bio-IgG) was preliminarily assembled with streptavidin based on biotin–streptavidin conjunction. Owing to four identical binding sites of the streptavidin to biotin, the amount of attached biotinylated alkaline phosphatase (bio-AP) increased to a certain extent. The attractive response performance of the developed electrochemical immunosensor strategy is presented and discussed in detail.

layer was firmly assembled on the electrode. After the blocking of nonspecific sites by BSA (0.25%, w/w), the immobilized antibody bound to corresponding antigen, which in turn sandwiched with another bio-IgG for the association of streptavidin. Subsequently, 3 ll of streptavidin (5 mg ml1) was dropped onto the modified electrode surface and incubated for 30 min in a moist environment for the immobilization of bio-AP. Finally, 10 ll of bio-AP was placed onto the electrode surface and incubated for 30 min. The AP enzymatic reaction progressed in 0.1 M Tris buffer (pH 9.0) containing 2 mM PAPP, 4 mM NADH, and 2 lM DI. All measurements were performed at room temperature. A schematic illustration of the stepwise self-assembled procedure of the immunosensor is shown in Fig. 1.

Materials and methods

Experimental measurements

Chemicals and materials

The stepwise modified process of electrodes was investigated by CV taking from 0.2 to 0.6 V (vs. SCE) at 50 mV s1 in 0.1 M PBS (pH 7.4) containing 5.0 mM FeðCNÞ4=3 and 0.1 M KCl at room 6 temperature. The electrochemical characteristics of the resulting immunosensors were investigated by CV taking from 0.3 to 0.4 V (vs. SCE) at 50 mV s1 in 0.1 M Tris buffer (pH 9.0) containing 2 mM PAPP, 4 mM NADH, and 2 lM DI. The electrochemical responses of the resulting immunosensors were proportional to the concentration of target human IgG.

Glutaraldehyde (GA), PAPP, bio-AP, DI (Bacillus stearothermophilus), NADH, titania nanoparticles (nano-TiO2), 3-aminopropyltrimethoxysilane (APTES), bovine serum albumin (BSA, 96–99%), and tris-hydroxymethylaminomethane hydrochloride (Tris) were purchased from Sigma (St. Louis, MO, USA). Human IgG, bio-IgG, streptavidin, and anti-IgG were obtained from Biocell Biotechnology (Zhengzhou, China). All other chemicals were of reagent grade and used as received. Tris–HCl buffer (0.1 M, pH 9.0) containing 1 mM MgCl2 was used to prepare enzyme solutions. Sodium phosphate buffer (PBS, 0.1 M) containing 10 mM KCl and 2 mM MgCl2 (pH 7.0) was used to prepare antibody and streptavidin solutions. PBS (0.1 M, pH 7.4) containing 5.0 mM FeðCNÞ4=3 and 0.1 M KCl was used 6 to investigate the assembled process of electrode. Double distilled water was used throughout this study. Apparatus Cyclic voltammetry was explored through a CHI 660A electrochemical workstation (Shanghai Chenhua Instrument, China). The pH measurements were made with a pH meter (MP 230, Mettler–Toledo, Switzerland). The electrochemical system consisted of a three-electrode system where bare or modified gold electrodes were used as a working electrode, platinum wire was used as an auxiliary electrode, and a saturated calomel was used as a reference electrode (SCE). Preparation of amino-functional nano-TiO2 The functionalization of TiO2 (NH2–TiO2) with APTES was prepared according to the literature [30] with slight modification. Briefly, l ml of acetic acid in methanol solution (1 mM) was prepared first, and then 500 ll of APTES and 300 ll of 5% TiO2 were added to the acetic acid and stirring was continued for 12 h. Subsequently, the resulting mixed solution was washed three times with methanol solution and finally dispersed in l ml of double distilled water. Fabrication of immunosensors Gold electrode (U = 4 mm) was carefully polished with 1.0 and 0.3 lm alumina powder separately. After rinsing with distilled water, the electrode was chemically cleaned by immersing into a freshly prepared H2SO4/H2O2 (2:1) mixture for 30 s. Then it was rinsed with distilled water and dried at room temperature. NH2–TiO2 (10 ll) was first assembled on the cleaned electrode surface to form a stable film with abundant –NH2 groups. With the help of the cross-linking agent GA (0.7%, w/w), anti-IgG mono-

Results and discussion Electrochemical characterization of stepwise modified electrodes The stepwise modified process was investigated in 0.1 M PBS (pH 7.4) containing 5.0 mM FeðCNÞ4=3 and 0.1 M KCl. Fig. 2 shows 6 the cyclic voltammograms of different electrodes in working buffer solution. The bare gold electrode had a couple of reversible redox peak (curve a). When the NH2–TiO2, GA, and anti-IgG films were formed on electrode surface successively, the electron transmission channel was hindered to a certain extent, leading to decreases in redox peaks (curves b–d). Subsequently, the immunosensor was blocked with BSA solution and then incubated in an incubation solution containing 1 ng ml1 IgG, and the redox peaks deceased further (curves e and f). The reasons may be ascribed to the electron transfer obstruction of the hydrophobic proteins on the surface of electrode. After being sandwiched with another bio-IgG for the association of streptavidin, the redox peaks deceased (curve g). Using the binding of biotin–streptavidin conjugates, streptavidin and bio-AP were assembled on the electrode surface, resulting in a decrease in redox peaks (curves h and i). To amplify electrochemical responses of the resulting immunosensors, the cyclic voltammograms of the characterized process and a multienzymatic system were employed. From Fig. 3, we can see that when the resulting immunosensor was characterized in 0.1 M Tris buffer (pH 9.0), there was no redox peak for the absence of electroactive compound (curve a). After the addition of electroinactive PAPP, a remarkable redox peak was obtained (curve b) and could be attributed to the hydrolysis of the PAPP substrate into the electroactive product PAP. In the presence of the auxiliary enzyme DI, the PAP was regenerated and the resulting oxidized form of DI was finally regenerated in its reduced native state by its natural substrate, NADH, which largely amplified the electrochemical responses (curve c). Optimization of experimental conditions for immunosensor The cyclic voltammograms at different scan rates in Fig. 4 show that both anodic and cathodic peak currents increased linearly

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Fig. 1. Schematic illustration of assembly process with the enzyme bioaffinity immunosensor.

200

20 i

Current / µA

100

Current / µA

a c e g

b d f h

0

-100

-200

c b

10

a

0

-10

-0.2

0.0

0.2

0.4

0.6

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Potential / V Fig. 2. Cyclic voltammograms of different electrodes obtained in 0.5 mM FeðCNÞ3=4 (pH 7.0): (a) bare Au; (b) TiO2/Au; (c) GA/TiO2/Au; (d) anti-IgG/GA/ 6 TiO2/Au; (e) BSA/anti-IgG/GA/TiO2/Au; (f) IgG/BSA/anti-IgG/GA/TiO2/Au; (g) bioIgG/IgG/BSA/anti-IgG/GA/TiO2/Au; (h) avidin/bio-IgG/IgG/BSA/anti-IgG/GA/TiO2/ Au; and (i) bio-AP/avidin/bio-IgG/IgG/BSA/anti-IgG/GA/TiO2/Au. The scan rate was 50 mV s1.

with the square root of scan rate between 10 and 250 mV s1, indicating a diffusion-controlled process of the APP/PQI couple at the modified electrode. As is known, the use of higher scan rates could bring about a value similar to that of the chemical system. However, a limiting factor was the fact that the definition of the peak current deteriorated simultaneously. Therefore, the scan rate of 50 mV s1 was adopted in our work. The amount of PAPP played a key role in the multienzymatic systems. When the amount of PAPP was not enough, a part of the AP on secondary antibody could not catalyze the PAPP hydrolysis for PAP sufficiently, and this had an influence on electrochemical response. Fig. 5 displays the properties of the resulting immunosensor in different concentrations of PAPP. The change of PAPP concentration was achieved by adding different volumes of high-concentration PAPP (48 mM) in 1 ml of working buffer solution. The current responses increased with the increase of PAPP

-0.2

0.0

0.2

0.4

Potential / V Fig. 3. Cyclic voltammograms of the resulting electrode obtained in 0.1 M Tris buffer (pH 9.0) (a); 0.1 M Tris buffer (pH 9.0) and 2 mM PAPP (b); and 0.1 M Tris buffer (pH 9.0), 2 mM PAPP, 4 mM NADH, and 2 M DI (c). The scan rate was 50 mV s1.

and then leveled off after adding 40 ll of 48 mM PAPP (equivalent to the concentration of PAPP in buffer solution reached to 2 mM). This meant that the amount of PAPP reached to saturation. Thus, PAPP at the concentration of 2 mM was selected for the optimum concentration of substrate that should be added in the immunoassay detection system. Nonspecific adsorption The nonspecific adsorption of proposed immunosensor has an influence on the achievement of a low detection limit. To investigate the nonspecific adsorption of immunosensors, we made a comparison that is shown in Fig. 6. Curve a shows that the cyclic voltammograms of electrode modified first with anti-IgG only had no obvious anodic peak current in the absence of AP. After the resulting immunosensor was incubated with 1014 g ml1 BSA (curve b) or 1014 g ml1 IgG (curve c), which in turn was sandwiched with bio-AP/streptavidin/bio-IgG, obvious anodic peak

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currents appeared. However, the electrochemical signal in curve c was nearly threefold larger than the one in curve b at a low target concentration, indicating that nonspecific adsorption of bio-IgG, bio-AP, and streptavidin was not significant.

60

250 10

20

Calibration curves of immunosensors 49

0 Current / µA

Current / µA

40

-20

r = 0.9921

42 35 28 21

-40

4

-0.4

-0.2

0.0

8 12 1/2 -1 1/2 V / (mV.s )

0.2

16

0.4

Potential / V Fig. 4. Cyclic voltammograms of the modified electrode at different scan rates (from inner to outer): 10, 20, 50, 80, 120, 150, 200, and 250 mV s1 in 0.1 M Tris buffer (pH 9.0) containing 2 mM PAPP, 4 mM NADH, and 2 M DI. The inset shows the dependence of redox peak currents on the potential sweep rates.

Under the optimized conditions, the performance of the current immunosensor was checked by detecting human IgG standard solutions with the CV technique in the presence of 2 mM PAPP, 4 mM NADH, and 2 lM DI in working buffer solution. As expected for a sandwich mechanism, the current response of PAP was proportional to the amount of enzyme conjugated to the second antibody, that is, proportional to the amount of targets to be detected. Here the nonspecific adsorption of the proposed immunosensor with different target concentrations was also investigated. Human IgG standard solutions were replaced by BSA of corresponding concentrations, and the responses are displayed by curve a in Fig. 7.

20

Current / µA

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30

12

Current / µA

Current / µA

18

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b

Current / µ A

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c a

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-0.4

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0.4

c

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0 -15

6

-30 -0.4

0 0

15

a -0.2

0.0 0.2 Potential / V

30

0 -16

0.4

45

-14

Volume / µl Fig. 5. The dependence of redox peak currents on the different concentrations of PAPP in 0.1 M Tris buffer (pH 9.0). The inset shows the corresponding cyclic voltammograms. The scan rate was 50 mV s1.

-12

-10

-8

-6

-4

-1 log[concentration] / g.ml

60

Fig. 7. The linear relationship between the changes of peak current and the target concentrations range from 1015 to 104 g ml1: (a) nonspecific adsorption of the resulting immunosensor; (b) resulting immunosensor in 2 mM PAPP, 4 mM NADH, and 2 M DI; and (c) resulting immunosensor in 2 mM PAPP. The inset shows the cyclic voltammograms of corresponding different resulting immunosensors incubated in 109 g ml1 BSA (a) or IgG (b and c).

14

7

25

b

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a

15 10

0

Current / µA

Current / µA

c

5

-7 -0.4

0

-0.2

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0.4

Potential / V Fig. 6. Cyclic voltammograms of different electrodes obtained in 0.1 M Tris buffer (pH 9.0) containing 2 mM PAPP, 4 mM NADH, and 2 M DI at a scan rate of 50 mV s1: (a) electrode modified with the first anti-IgG only; (b) resulting immunosensor incubated with 10–14 g ml1 BSA; and (c) resulting immunosensor incubated with 10–14 g ml1 IgG.

IgG ne i yste G L-c HC

Ta

rge

t

3 2 BSA

P

AF

1

n

Fig. 8. Experiment of interference with the proposed immunosensor (with n devoted to the regeneration times).

Electrochemical enzyme bioaffinity electrode / Y. Yuan et al. / Anal. Biochem. 405 (2010) 121–126

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Table 1 Reproducibility assays using five equally prepared electrodes.

CIgG (ng ml1)a a

Electrode 1

Electrode 2

Electrode 3

Electrode 4

Electrode 5

0.92 ± 0.02

0.98 ± 0.04

1.05 ± 0.05

1.02 ± 0.03

1.04 ± 0.02

Calculated as a mean of three measurements.

There were low electrochemical signals, indicating no significant nonspecific adsorption of the proposed immunosensor. Subsequently, the calibration plots of the anodal peak current responses changes via concentrations of human IgG standard solution are illustrated by curve b. With the dual-amplification effect of multienzyme catalysis toward the presence of PAPP, the linear response to human IgG ranged from 1014 to 105 g ml1 with a relatively low detection limit of 3.5  1015 g ml1 (3r). For comparison, the current responses of the proposed immunosensor were recorded without the addition of NADH and DI and are depicted by curve c. The electrochemical responses decreased, and the immunosensors showed linear ranges of 1013–105 g ml1. The phenomenon showed that the multienzymatic systems could provide a possible enhancement of electrochemical signals, enlargement of linear range, and improvement of sensitivity, as expected. Specificity, stability, and repeatability To investigate the specificity of the proposed immunosensor for IgG, some potential interferents, such as a-fetoprotein (AFP), BSA, L-cysteine, and human chorionic gonadotropin (HCG), were used to evaluate selectivity of the immunosensor. From Fig. 8, we can see that only the proposed immunosensor incubated in IgG (109 g ml1) had obvious anodal peak current responses, and this validated the high specificity of the proposed immunosensor. The stabilities of the proposed immunosensor were researched by successive cycle scan and long-term storage assay. Under the optimal conditions at 50 mV s1, the electrode lost only 7.5% of its initial response after approximately 100 continuous measurements. On the other hand, the long-term storage stability was investigated over a period of 20 days of storage (at 4 °C) and occasional running. The CV peak current of the immunosensor decreased gradually and retained 90.4% of its initial current after the first 10 days of storage and 84.6% after 20 days of storage. These results indicated that the stability of the immunosensor was satisfactory in this experiment. The reproducibility of the immunosensor was investigated by analysis of the same concentration of IgG (109 g ml1) with five equally prepared electrodes. The results are shown in Table 1, and an acceptable relative standard deviation of 5.30% (n = 5) was calculated. Conclusions An electrochemical immunosensor with high sensitivity and a low detection limit has been successfully demonstrated in this work. Two main factors could be ascribed to the high sensitivity. One was the employment of streptavidin, which brought about more bio-AP conjugated on the electrode surface, leading to an increase in catalytic ability of the proposed immunosensor. The other one was the introduction of two enzymatic amplifiers: DI and NADH. Here the auxiliary enzyme DI made a reversible cycle of PAP, and the oxidized form of DI was regenerated in its reduced native state by its natural substrate, NADH, forming a bienzyme substrate recycling for dual signal amplification of electrochemical signals. The results described here are promising because of the immunosensor’s high sensitivity, low detection limit, and wide detection range.

Acknowledgments This work was supported by the National Natural Science Foundation of China (20675064), the Natural Science Foundation of Chongqing City (CSTC-2009BA1003), the Ministry of Education of China (Project 708073), the High Technology Project Foundation of Southwest University, China (XSGX02), and the Doctor Foundation of Southwest University (SWU109016). References [1] D.P. Tang, R. Yuan, Y.Q. Chai, Biochemical and immunochemical characterization of the antigen–antibody reaction on a non-toxic biomimetic interface immobilized red blood cells of crucian carp and gold nanoparticles, Biosens. Bioelectron. 22 (2007) 1116–1120. [2] G.S. Lai, F. Yan, H.X. Ju, Dual signal amplification of glucose oxidasefunctionalized nanocomposites as a trace label for ultrasensitive simultaneous multiplexed electrochemical detection of tumor markers, Anal. Chem. 81 (2009) 9730–9736. [3] V. Mani, B.V. Chikkaveeraiah, V. Patel, J.S. Gutkind, J.F. Rusling, Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification, ACS Nano 3 (2009) 585– 594. [4] D. P Tang, J.J. Ren, In situ amplified electrochemical immunoassay for cow microspheres as labels, Anal. Chem. 80 (2008) 8064–8070. [5] R.J. Cui, H.C. Pan, J.J. Zhu, H.Y. Chen, Versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels, Anal. Chem. 79 (2007) 8494–8501. [6] G.D. Liu, Y.Y. Lin, J. Wang, H. Wu, C.M. Wai, Y.H. Lin, Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip, Anal. Chem. 79 (2007) 7644–7653. [7] Y. Higashi, Y. Ikeda, R. Yamamoto, M. Yamashiro, Y. Fujii, Pharmacokinetic interaction with digoxin and glucocorticoids in rats detected by radioimmunoassay using a novel specific antiserum, Life Sci. 77 (2005) 1055– 1067. [8] C.Y. Chan, Y. Bruemmel, M. Seydack, K.K. Sin, L.W. Wong, E. Merisko-Liversidge, D. Trau, R. Renneberg, Nanocrystal biolabels with releasable fluorophores for immunoassays, Anal. Chem. 76 (2004) 3638–3645. [9] M.S. Wilson, Electrochemical immunosensors for the simultaneous detection of two tumor markers, Anal. Chem. 77 (2005) 1496–1502. [10] M.S. Wilson, W. Nie, Multiplex measurement of seven tumor markers using an electrochemical protein chip, Anal. Chem. 78 (2006) 6476–6483. [11] Y.F. Wu, C.L. Chen, S.Q. Liu, Enzyme-functionalized silica nanoparticles as sensitive labels in biosensing, Anal. Chem. 81 (2009) 1600–1607. [12] J. Das, K. Jo, J.W. Lee, H. Yang, Electrochemical immunosensor using paminophenol redox cycling by hydrazine combined with a low background current, Anal. Chem. 79 (2007) 2790–2796. [13] A. Warsinke, A. Benkert, F.W. Scheller, J. Fresenius, Electrochemical immunoassays, Anal. Chem. 366 (2000) 622–634. [14] D. Knopp, Immunoassay development for environmental analysis, Anal. Bioanal. Chem. 385 (2006) 425–427. [15] H. Dong, C.M. Li, W. Chen, Q. Zhou, Z.X. Zeng, J.H.T. Luong, Sensitive amperometric immunosensing using polypyrrolepropylic acid films for biomolecule immobilization, Anal. Chem. 78 (2006) 7424–7431. [16] M.S. Wilson, W.Y. Nie, Electrochemical multianalyte immunoassays using an array-based sensor, Anal. Chem. 78 (2006) 2507–2513. [17] A. Preechaworapun, T.A. Ivandini, A. Suzuki, A. Fujishima, O. Chailapakul, Y. Einaga, Development of amperometric immunosensor using boron-doped diamond with poly(o-aminobenzoic acid), Anal. Chem. 80 (2008) 2077– 2083. [18] Z.P. Aguilar, I. Fritsch, Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum Hsp70 mRNA, Anal. Chem. 75 (2003) 3890–3897. [19] Z.P. Aguilar, Small-volume detection of Plasmodium falciparum CSP gene using a 50-(m-diameter cavity with self-contained electrochemistry, Anal. Chem. 78 (2006) 1122–1129. [20] Z.P. Aguilar, W.R. Vandaveer, I. Fritsch, Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system, Anal. Chem. 74 (2002) 3321–3329. [21] Y. Wang, M. Stanzel, W. Gumbrecht, M. Humenik, M. Sprintzl, Esterase 2oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization, Biosens. Bioelectron. 22 (2007) 1798– 1806.

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