Electrochemical amperometric immunoassay for carcinoembryonic antigen based on bi-layer nano-Au and nickel hexacyanoferrates nanoparticles modified glassy carbon electrode

Journal of Electroanalytical Chemistry 626 (2009) 6–13

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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem

Electrochemical amperometric immunoassay for carcinoembryonic antigen based on bi-layer nano-Au and nickel hexacyanoferrates nanoparticles modified glassy carbon electrode Yan-Ru Yuan, Ruo Yuan *, Ya-Qin Chai, Ying Zhuo, Xiang-Min Miao Chongqing Key Laboratory of Analytical Chemistry, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

a r t i c l e

i n f o

Article history: Received 15 May 2008 Received in revised form 13 October 2008 Accepted 16 October 2008 Available online 20 November 2008 Keywords: Amperometric immunosensor Carcinoembryonic antigen Nano-Au Hexacyanoferrates nanoparticles

a b s t r a c t This study demonstrates a new approach towards development of novel immunosensor based on gold nanoparticles (nano-Au) and nickel hexacyanoferrates nanoparticles (NiHCFNPs) for determination of carcinoembryonic antigen (CEA) in clinical immunoassay. The fabrication steps of the immunosensor as follows: firstly, nano-Au was immobilized on the surface of bare glassy carbon electrode (GCE) by using a simple method – electrochemical reduction of HAuCl4 solution; secondly, NiHCFNPs as an electroactive substance was immobilized on the layer of gold nanoparticles. Microstructure and surface morphology of NiHCFNPs have been characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); thirdly, nano-Au was again immobilized on the surface of NiHCFNPs, which can offer a favorable microenvironment and biocompatibility to immobilize anti-CEA. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to characterize the electrochemical properties of modified process. Effect of deposition time of nano-Au, pH of working buffer, incubation temperature and time were studied in detail for optimization of analytical performance. Under optimal conditions, the peak current of CV of the immunosensor decreased linearly with increasing CEA concentration in two ranges from 0.5 to 10.0 ng mL1 and from 10.0 to 160.0 ng mL1, with a detection limit 0.1 ng mL1 at three times background noise. The proposed immunosensor show good repeatability and reproducibility, acceptable accuracy, high sensitivity and would be valuable for diagnosis and monitoring of carcinoma. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction In the tumor process, increased level of tumor markers in human serum, are associated with certain tumor. Therefore, determination of tumor markers, potential prognostic factors for tumors, plays an important role in clinical research and diagnosis [1,2]. Carcinoembryonic antigen (CEA), is typically associated with certain tumors and the developing fetus [3,4] and is widely used as clinical tumor marker for some familiar cancers [5–8]. The normal range for CEA in an adult non-smoker is <2.5 ng mL1 and for a smoker <5.0 ng mL1. A rising CEA level indicates progression or recurrence of the cancer [9,10]. Thus, the detection of CEA levels in human serum is necessary in clinical assay. The technique based on high specific molecular recognition of antigen by antibody and used for quantitative determination of tumor markers is usually immunoassay. Many kinds of immunoassay methods [11–17], have been applied for the detection of CEA. However, methods described above have always the disadvantages, * Corresponding author. Tel.: +86 23 68252277; fax: +86 23 68254000. E-mail address: [email protected] (Y.-R. Yuan). 0022-0728/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2008.10.031

such as time consuming, requiring highly qualified personnel, sophisticated instrumentation, poor precision, difficult to realize automation [18,19]. Electrochemical immunoassay combining the features of fast analysis, sensitive and precise measurement, simple pretreatment, inexpensive and miniaturizable instrumentation, has drown more attention in a wide range of uses [20]. Electrochemical amperometric immunoassay with amperometric transducer is receiving intense attention because it can achieve a relatively low detection limit and high sensitivity. The amperometric immunoassay also plays an increasing role in immunosensors. Thus, looking for a novel immobilization method for amperometric immunosensor with great improvement in sensitivity, selectivity, and response time is of considerable interest. Advanced material based on inorganic and organic nanoparticles is nowadays one of the key research fields of today material science. Among these nanomaterials, noble metal nanoparticles have been used to fabricate biosensor owing to their excellent properties to immobilize biomolecules (such as enzyme, antibody, antigen, DNA, RNA) [21–24]. The nano-Au has drawn much attention in constructing electrical and optical sensors due to their small size and correspondingly unique optical, electronic,

Y.-R. Yuan et al. / Journal of Electroanalytical Chemistry 626 (2009) 6–13

and catalysis properties. The nano-Au not only can offer a microenvironment similar to nature and retain the bioactivity of the immobilized biomolecules, but also can provide a high surface to volume ratio and enhance the electron transfer kinetics by giving more freedom energy to the immobilized biomolecular in orientation which makes active sites closer to conducting electrode and permit the biomolecules to orient in conformation more favorable for direct electron transfer [25,26]. In our present work, a nano-Au layer on the electrode was prepared by using a simple method – electrochemical reduction of HAuCl4 solution that agrees with literatures [27–29]. The nano-Au film formed by this method can be achieved in a relatively short time and can provide a stable and rough surface that is more favorable to immobilize biomolecules. Metal hexacyanoferrate complexes (MHCFs) modified electrodes have attracted considerable interest due to their potential application in many fields [30,31]. Among PB analogues, nickel hexacyanoferrate exhibits attractive redox properties in term of charge compensation [32]. In addition, it has been to the electrocatalysis and determination of organic and inorganic species [33]. So far, little attention has paid to use nickel hexacyanoferrate nanoparticles (NiHCFNPs) to construct biosensors. NiHCFNPs is of lager surface area, a high surface to volume ratio, and high affinity to nano-Au due to existing strong interaction between – NH2, –SH, –CN, and nano-Au [34–37]. It will be an important improvement to modify NiHCFNPs onto nano-Au layer for biosensing application. In the present work, NiHCFNPs was synthesized by simply mixing nickel ions and hexacyanoferrate (K3Fe(CN)6) at room temperature. After that, NiHCFNPs as a good electroactive substance at the same time was immobilized on the electrode, which greatly simplifies the immunoassay system. However, many amperometric immunoassay techniques need an electroactive substance to analytical system, which lead to more complex immunoassay system and increased analytical time and expense [38,39]. Recently, we also developed some electrochemical immunosensors based on nanomaterials with favorable biocompatibility for the determination of CEA [40–42]. Sensitivities of the immunosensors need to be improved. In the present investigation, we simultaneously took advantages of two kinds of nanomaterial (nanoAu and NiHCFNPs) with good properties, tried to develop a simple and sensitive immobilization strategy for constructing a compatible amperometric immunoassay. At first, nano-Au layer with favorable biocompatibility and large surface area was formed by simple electrochemical reduction method. Secondly, NiHCFNPs as a good electroactive substance was self-assembled on nano-Au layer by strong interaction between CN (NiHCFNPs) and nanoAu [34–37]. Thus, the second nano-Au layer was again self-assembled onto NiHCFNPs by simply electrochemical reduction, which offers a biocompatible interface to adsorb anti-CEA by chemical adsorption between nano-Au and NH2 of anti-CEA. In addition, nano-Au layer formed by this method can offer a stable, rough and compact surface, which can immobilize more amount antibody. There are two novel strategies in designing the immunosensor as follows: First, promising polymeric inorganic compound NiHCFNPs was not only an excellent electroactive substance, but also could provide a favorable interface for nano-Au immobilization, which greatly improve performance of immunosensor. The nano-Au and NiHCFNPs both with excellent properties were simultaneously used to design an accuracy, sensitivity and stability immunosensor. Second, the preparation of immunosensor is simple, easy controlled and less time consuming. The performance and factors influencing the immunosensor’s performance were also investigated in detail. The proposed immunosensor was applied to determine CEA in human serum samples with satisfactory results.

7

2. Experimental 2.1. Reagents and materials CEA and anti-CEA (Biocell Company, Zhengzhou, China), Bovine serum albumin (BSA, 96–99%), gold chloride (HAuCl4) and sodium citrate (Sigma, St. Louis, MO, USA), K3Fe(CN)6 and NiCl26H2O (Chemical Reagent Co, Sichuan, China). All other chemicals and solvents (analytical grade, regular sources), Bi-distilled water was employed throughout this study. Phosphate buffered solutions (PBS) at various pH were prepared using 0.02 M Na2HPO4 and 0.02 M KH2PO4 stock solution. The supporting electrolyte was 0.1 M KCl. The CEA was stored in the frozen state, and its standard solutions were prepared freshly with bi-distilled water when in use. 2.2. Apparatus CHI 610A electrochemistry workstation (Shanghai CH Instruments, China), Model IM6e (ZAHNER Elektrick, Germany), pH meter and digital ion analyzer (Model PHS-3C, DaPu Instruments, Shanghai, China), transmission electron microscopy (TEM) (TECNAI 10, PHILIPS, Holand), S-3400 scanning electron microscope (SEM) (Hitashi High Technologies Corporation, Japan). 2.3. Preparation of nickel hexacyanoferrates nanoparticles (NiHCFNPs) The synthesis of NiHCFNPs was according to the literature [43] with a little modification: 70 mL of 0.01 M NiCl2 aqueous solution was drop by drop added to 70 mL of 0.05 M K3Fe(CN)6 aqueous solution containing 0.05 M KCl under stirring. After finished addition, the mixture solution was vigorously agitated for 5 min. Following that, the mixture solution was immediately centrifuged, and washed with bi-distilled water for several times. Then dried NiHCFNPs overnight in a vacuum at room temperature and finally gave a powered substance. 2.4. Preparation of the immunosensor The glassy carbon electrode (GCE) (U = 4 mm) was first polished to a mirror finish respectively with 1.0, 0.3 lm alumina slurry, followed by rinsing thoroughly with bi-distilled water after each polishing step. After that, the electrodes were successively sonicated in 1:1 nitric acid, ethanol, bi-distilled water. Following dried in air. The electrode was modified immediately after the cleaning steps. The preparation of nano-Au layer was constructed by immersed the cleaning electrode in HAuCl4 aqueous solution (2 mg mL1) and applied constant potential 0.2 V for 60 s. It was rinsed with a copious amount of water and a yellow nanoAu layer can be seen. Before the step of modification, 0.05 M NiHCFNPs aqueous solution need be prepared firstly. When NiHCFNPs was dispersed in bidistilled water, its aqueous solution was very stable and no precipitate was observed after 2 months when stored at 4 °C. A 10 lL NiHCFNPs aqueous solution (0.05 M) was dropped onto the surface of nano-Au layer formed right now. Immediately transfer the electrode to refrigerator and keep at 4 °C for about 2 h for NiHCFNPs film dry. When NiHCFNPs film dried, the electrode was washed thoroughly with bi-distilled water. The NiHCFNPs/nano-Au modified electrode was soaked in 3 mL HAuCl4 aqueous solution (2 mg mL1) and applied constant potential 0.2 V for 30 s. Following that, the modified electrode washed with bi-distilled water. Subsequently, the nano-Au/NiHCFNPs/ nano-Au modified electrode was immersed in anti-CEA solution at 4 °C for about 12 h. Finally, the proposed electrode was incubated

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Scheme 1. Schematic diagram of the stepwise immunosensor construction process: (a) electrochemical reduction formation of nano-Au; (b) assembled the NiHCFNPs; (c) formation of nano-Au film; (d) anti-CEA loading; and (e) blocked nonspecific sites with BSA.

in BSA for 1 h at 37 °C. The accomplished immunosensor was stored at 4 °C when not in use. The schematic diagram of the stepwise self-assemble procedure of the immunosensor was shown in Scheme 1. 2.5. Experiment measurement The electrochemical impedance technique is employed to detect impedance change and thickness of surface of the electrode during self-assemble (SA) process. The stepwise immunosensor fabrication processes were characterized by using electrochemical impedance measurements (EIS). It was carried out in the presence 3 as a redox probe. of a 5 mM FeðCNÞ4 6 = Electrochemical experiments were performed in a conventional three-electrode electrochemical cell. A three-electrode electrochemical cell contained a platinum wire auxiliary electrode, a saturated calomel reference electrode (SCE) and the modified glassy carbon electrode (GCE) (U = 4 mm) as working electrode. The CV measurement was taken from 0.0 to 0.8 V (vs. SCE) at 50 mV s1 in 0.02 M PBS (pH = 7.0) at room temperature. The amperometric detection is based on the change in the amperometric response (4ip) before and after antigen-antibody reaction. When the antibody immobilized on the electrode has bound with antigen, the complex of antigen–antibody coating on the surface of the electrode acts as inert substance and badly blocks the electron transfer tunnel to the electrode surface. Moreover, with increasing the concentration of antigen, the amount of antigen–antibody complex also increases, which result in less tunnels of electron transfer and the decrease of current signal of electroactive substance. Meantime, the peaks current signal of electroactive substance decrease directly proportional to the concentration of antigen that has reacted with antibody immobilized on the electrode surface.

Fig. 1. The TEM images of nickel hexacyanoferrates nanoparticles (NiHCFNPs).

was about 10 nm. A relatively uniform structure appeared in TEM of NiHCFNPs. Moreover, the surface morphology of nano-Au, NiHCFNPs and anti-CEA on the electrode was characterized with SEM. As shown in Fig. 2a, TEM of nano-Au photograph displayed a chemically relative congregate surface microstructure, and the size of nano-Au is about 200 nm. The aggregates of NiHCFNPs on the electrode surface showed a very narrow particle size distribution (Fig. 2b); when gold nanoparticles were again immobilized on the surface of NiHCFNPs, a relatively uniform distribution of nanoAu was obtained (Fig. 2c). Fig. 2d showed images of anti-CEA/nanoAu/NiHCFNPs/nano-Au. Big biomolecule anti-CEA displayed hill like microstructure.

3. Results and discussion

3.2. Electrochemical characterization of the immunosensor

3.1. Morphology of NiHCFNPs

CV is an effective and convenient method for probing the feature of the modified electrode surface. Thus, CV was selected as a marker to investigate electrochemical behavior after each assembly step. CVs of the different modified stepwise of immunosensor are shown in Fig. 3. No peak was observed at the bare GCE as the lack of electroactive substance (Fig. 3a). It can be observed a little

The morphology of NiHCFNPs is an important factor affecting the performance of an immunosensor. TEM is an effective method to provide the information on particle size and shape. Fig. 1 exhibited the microstructure of NiHCFNPs. The average size of NiHCFNPs

Y.-R. Yuan et al. / Journal of Electroanalytical Chemistry 626 (2009) 6–13

9

Fig. 2. The typical SEM images of (a) nano-Au modified surface obtained by electrochemical reduction of HAuCl4 solution; (b) NiHCFNPs/nano-Au surface; and (c) nano-Au/ NiHCFNPs/nano-Au surface; (d) anti-CEA/nano-Au/ NiHCFNPs/nano-Au surface.

300

I/µA

d

200

c

100

e f

0 -100

g b

a

a

-200 -300 0.0

0.2

0.4 E/V

0.6

0.8

Fig. 3. Cyclic voltammograms of the different electrodes measured in 0.02 M PBS (pH7.0) at 25 °C: (a) bare GCE; (b) nano-Au modified electrode; (c) NiHCFNPs/nanoAu modified electrode; (d) nano-Au/NiHCFNPs/nano-Au modified electrode; (e) anti-CEA/nano-Au/NiHCFNPs/nano-Au modified electrode; (f) BSA/ anti-CEA/ nanoAu/NiHCFNPs/nano-Au modified electrode (the resulting immunosensor); and (g) the resulting immunosensor incubated in 20.0 ng mL1 CEA. The potential scan rate was 50 mV s1.

increase in background current due to increase of the double layer capacitance from nano-Au modification (Fig. 3b). However, after NiHCFNPs was modified onto the nano-Au layer, peak current in-

creased greatly and appeared a well defined CV, which can attribute to NiHCFNPs be an excellent electroactive substance and favorably enhance transfer of the electron (Fig. 3c). When the NiHCFNPs/nano-Au modified electrode was further modified by the nano-Au, the peak current increased a lot owing to noble metal Au nanoparticles layer could form more electron transfer tunnels that facilitated electron transmission (Fig. 3d). Peak current decreased clearly after the anti-CEA was adsorbed on the nano-Au layer (Fig. 3e), which suggested the big molecular protein antiCEA severely blocks the electron transfer tunnel and reduced effective area of electron transfer. Subsequently, when BSA was used to block nonspecific sites, the peak current further decreased (Fig. 3f). The reason may be that the big biomolecular BSA hinders the tunnel of electron transfer to the electrode surface. After the immunosensor incubated in 20.0 ng mL1 of CEA for 20 min, a dramatically decrease in current is observed (Fig. 3g), that contribute to the formation of immunocomplex retardation of the electron transfer tunnel. The CVs of the resulting immunosensor incubated in 20.0 ng mL1 CEA in 0.02 M PBS at different scan rates ranging from 20 mV s1 to 250 mV s1 was investigated. As Fig. 4 shown, the potential and peak currents are dependent on the scan rate. It can be also observed that the redox peak currents were proportional to the scan rate, shown in the inset, suggesting that a surface limited process. Moreover, the impedance changes of immunosensor surface in the fabrication process and the formation of antigen–antibody complex can be observed by EIS. Thus, the stepwise construction process of the immunosensor was characterized by EIS. Fig. 5a,

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Y.-R. Yuan et al. / Journal of Electroanalytical Chemistry 626 (2009) 6–13

I/µA

200

(Fig. 5e). The result is consistent with the fact that the hydrophobic layer of protein insults the conductive support and hinders the interfacial electron transfer. Ret increased in the same way after BSA was used to block nonspecific sites (Fig. 5f), which may attribute to the same reason with loading the antibody. After the resulting immunosensor was incubated in 20.0 ng mL1 of CEA, Ret further increases (Fig. 5g), which indicates the formation of hydrophobic immunocomplex layer embarrassing the electron transfer.

I/µA

100 v

0

3.3. Optimization of analytical conditions for the immunoassay

-100

-200

0.0

0.2

0.4

0.6

0.8

E/V Fig. 4. CVs of the immunosensor incubated in 20 ng mL1 CEA solution at various scan rates in 0.02 M PBS (pH = 7.0) at 25 °C. Scan rates from inner to outer: 20, 50, 80, 100, 120, 150, 180, 200, and 250 mV s1. The inset shows the dependence of the redox peak currents on scan rates.

-1200 -1000

zim/ohm

-800 -600

d

-400

b

a

f

e

c

g

-200 0

0

200

400

600

800

1000

1200

1400

1600

Zre/ohm Fig. 5. Electrochemical impedance spectroscopy (EIS) of the different electrodes 3 at 25 °C: (a) bare GCE; (b) nano-Au modified measured in 5 mM FeðCNÞ4 6 = electrode; (c) NiHCFNPs/nano-Au modified electrode; (d) nano-Au/NiHCFNPs/nanoAu modified electrode; (e) anti-CEA/ nano-Au/NiHCFNPs/nano-Au modified electrode; (f) BSA/anti-CEA/ nano-Au/NiHCFNPs/nano-Au modified electrode (the resulting immunosensor); and (g) the resulting immunosensor incubated in 20.0 ng mL1 CEA.

showed EIS of the bare GCE. A very small semicircle at high frequencies and linear part at low frequencies was observed, which agrees with literature [44], suggesting very low Ret to redox probe 3 FeðCNÞ4 6 = . After the bare electrode was modified by nano-Au layer (Fig. 5b), the EIS exhibits a very low interfacial Ret, indicating the resulting nano-Au layer greatly enhanced electron transfer of the redox probe. When NiHCFNPs again modified the nano-Au/ GCE, it can be found that interfacial Ret increases but less than that of bare GCE (Fig. 5c). The reason may be that the microstructure of NiHCFNPs gives birth to some barrier obstructing the electron transfer. Then the Ret decreases obviously when nano-Au was again assembled on the NiHCFNPs (Fig. 5d), which maybe ascribed to nano-Au be a kind of conductive material and its conductivity be better than that of NiHCFNPs. Subsequently, when the anti-CEA was loaded on the surface of nano-Au, Ret increases dramatically

3.3.1. Reduction time of nano-Au Time of electrochemical reduction HAuCl4 has great effect on performance. Less reduction time, nano-Au layer formed is very thin and amount of nano-Au is few, which directly affect the immobilization of NiHCFNPs and further influence performance of the immunosensor. The more reduction time, the more amount of nano-Au, but if reduction time exceeding some value might result in excessive compactness film of Au particles, which seriously obstructs electron transfer tunnels and further affected the response of the immunosensor. The effect of first nano-Au layer 3 solution when reduction time ranwas researched in FeðCNÞ4 6 = ged from 20 s to 100 s. As Fig. 6a shown, at 60 s, the current response reached a maximum value. A 60 s was chosen as the reduction time of first nano-Au layer. Moreover, the reduction time of second nano-Au layer also need to be optimized. The influence of second nano-Au layer was studied in 0.02 M PBS with the reduction time ranged from 10 s to 60 s. Why a 0.02 M PBS was selected as characterization solution? The reason might be that before modified the second nano-Au layer, NiHCFNPs as an excellent electroactive substance had been assembled on the nano-Au/GCE, which offered enough sensitivity to optimize the reduction time of second nano-Au layer. The result shows that the reduction time of second nano-Au layer arrive at maximum response at 30 s (Fig. 6b). Thus 30 s was used as the reduction time of second nano-Au layer. 3.3.2. pH of working buffer The effect of pH on the immunosensor behavior was investigated between 5.0 and 9.0 in 0.02 M PBS. As shown in Fig. 7, the peak current increases with increasing pH value from 5.0 to 7.0 and decreases when further increases pH value. The test results show that the maximum current response occurs at pH 7.0. So a pH 7.0 PBS is chosen throughout this study. 3.3.3. Incubation temperature and time To our knowledge, the optimal temperature of immunoreaction is 37 °C. It is also the temperature of human being. But taking consideration of the activity of biomolecules and life-time of immunosensor, 25 °C was selected as incubation temperature for whole assays. At this temperature, the immunosensor was incubated in a standard solution of CEA with known constant concentration of 20.0 ng mL1 for different time. As Fig. 8 shown, the response current of immunosensor was rapidly up with the duration of incubation time from 2 min to 20 min, and then leveled off slowly, which indicated an equilibrium state reached. Thus, 20 min was used for incubation time of all the subsequent assays. 3.4. Performance of immunosensor 3.4.1. Amperometry response of the proposed immunosensor After the immunosensor was incubated in a standard CEA solution with constant concentration 20.0 ng mL1 for 20 min at 25 °C, the amperometry response can be obtained in 0.02 M PBS (pH = 7.0). As shown in Fig. 3g, peak current decreased as expected; correspondingly, in Fig. 5g, the Ret increased remarkably. When the

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Y.-R. Yuan et al. / Journal of Electroanalytical Chemistry 626 (2009) 6–13

a

b

160

174

171

156

I/µA

I/µA

168

165

152

162

148 159

40

20

60

80

100

10

20

t/sec

30

40

50

60

t/sec

Fig. 6. The effect of electrochemical reduction time of nano-Au on response currents of modified electrode characterized by CV in different solution at 25 °C: (a) the electrode 3 ; (b) the electrode nano-Au/NiHCFNPs/nano-Au in 0.02 M PBS (pH = 7.0). The potential scan rate was 50 mV s1. of nano-Au/GCE in 5 mM FeðCNÞ4 6 =

-68 112

-72

I/µA

I/µA

105

98

-76

-80 91

-84 84 5

6

7

8

9

pH Fig. 7. The influence of pH of the PBS on the response of the immunosensor in 0.02 M PBS at 25 °C. The scan rate was 50 mV s1.

proposed immunosensor was incubated in standard solution of CEA with different concentration with other conditions the same as described above, the calibration plot of the determination of CEA can be gained along with obtaining CVs of different concentration of CEA. Fig. 9 illustrates that the calibration plot for the detection of CEA. The amperometry response linearly increased with the increase of CEA concentration in two ranges from 0.5 to 10.0 ng mL1 and from 10.0 to 160.0 ng mL1, with a detection limit 0.1 ng mL1 at a signal to noise ratio of 3. The linear slopes were 2.6696 and 0.1704 lA (ng mL1)1, and the correlation coefficients were 0.9874 and 0.9955, respectively. Why calibration curve presented two slopes? The reason may be: the amount of antibody immobilized on the immunosensor is some certain. When the immunosensor is used to detect antigen, antigen can bind with active sites of antibody which was immobilized on the immunosensor. With increase of antigen concentration, active sites of antibody on the immunosensor become fewer and fewer. As a result, when the immunosensor is used to determine higher concentration of antigen, the sensitivity can decline. There are literatures [45–50] whose calibration curves refer two slopes. The proposed method is thus absolutely means to quantify CEA in the sample, which indi-

0

5

10

15

20

25

Time/min Fig. 8. The effects of incubation incubation time; CV determination was performed in 0.02 M PBS (pH = 7.0) at 25 °C. The scan rate was 50 mV s1.

cated that this immunosensor has great potential to be used in clinical diagnosis. 3.4.2. Selectivity against interferences Selective determination of target analytes plays an important role in analyzing biological samples in situ without separation. The effect of substances that might interfere with the response of immunosensor was investigated. The evaluation selectivity of the immunosensor was carried out by incubated the immunosensor in 20.0 ng mL1 CEA containing some potential co-existed species with CEA, such as alpha-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), human chorionic gonadotrophin (HCG), BSA, ascorbic acid, dopamine, L-glucose, L-cystein, L-lysine, serine, arginine, histidine, leucine. The degree of interference from substances described above can be judged from the value of the current ratio. In this study, the current ratios can be obtained from the current reading of the immunosensor in 0.02 M PBS (pH = 7.0) with the immunosensor incubated in 20.0 ng mL1 CEA and 20.0 ng mL1 interfering substances versus the current reading with the immunosensor incubated in 20.0 ng mL1 CEA. The test results were

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Y.-R. Yuan et al. / Journal of Electroanalytical Chemistry 626 (2009) 6–13 Table 2 Comparison of serum levels by using two methods.

-30

Serum samples

-40

1

This method (ng mL ) CLIA (ng mL1) Relative deviation (%)

I/µA

-50 -60

1

2

3

4

5

6

1.8 1.7 5.9

4.6 4.3 7.0

8.3 8.5 2.4

15.7 16.4 4.3

29.2 28.6 2.1

56.4 58.3 3.3

Table 3 Comparision of the performance of electrochemical immunosensors for the determination of CFA.

-70 -80 -90 0

30

60

90

120

150

180

-1

C/(ng mL ) Fig. 9. Calibration plot of the immunosensor for detection of CEA. The amperometry measurement was carried out by CVs in 5 mL 0.02 M PBS (pH = 7.0) at 25 °C. The scan rate was 50 mV s1.

listed in Table 1. The results suggested that the substance selected did not interfere remarkably. However, these non-specific species might result in current shift or current change in certain error range. 3.4.3. Stability, repeatability, and reproducibility The successive stability of the immunosesor was researched. After the imunosensor was successively scanned for 80 circles in working buffer, a RSD of 4.3% was gained. The immunosensor had acceptable storage stability with 89% and 75% of initial response remaining after the storage periods of 14 days and 30 days in 0.02 M PBS (pH = 7.0) at 4 °C. When the immunosensor was measured for 11 times in working buffer, respectively, it yielded a 1.9% RSD. The reproducibility of immunosensors plays a key factor for developing a practical immunosensor. A regeneration study of the prepared immunosensor was implemented by using regeneration solutions to break the antibody–antigen linkage. In this study, the two different immunosensors that had been incubated in 20.0 ng mL1 CEA for 20 min, were immersed in a stirred 0.2 M glycine-hydrochloric acid (Gly-HCl) solution (pH = 2.8) and a stirred 4 M urea solution for 5 min, respectively. RSD of 4.7% and 6.2% was acquired, respectively (4 regenerations and measurement). 3.4.4. Preliminary analysis of real human serum samples To demonstrate the application of the proposed immunosensor for the detection of the CEA in human serum, six serum samples with different infection degrees from the Ninth people’s Hospital of Chongqing, were examined by the proposed electrochemical immunoassay and chemiluminescene immunoassay (CLIA). Con-

Table 1 Possible interferences tested with the proposed electrochemical immunosensor. Possible interferences

Current ratio

Possible interferences

Current ratio

Alpha-fetoprotein Hepatitis B surface antigen Human chorionic gonadotrophin Bovin serum antigen L-Glutamate Serine Arginine

0.89 0.92 0.93

L-Cysteine

Histidine

1.04 0.96 1.03

Leucine Ascorbic acid Dopamine

1.01 0.94 0.95

1.05 1.03 1.02 0.98

L-Lysine

Type of immunosensor

Linear ranges (ng mL1)

Electrochemical

0.5–3.0 and 3.0– 120 25–150 0.5–25 0.5–80.0 3.0–50

Electrochemical Electrochemical Electrochemical Quartz crystal microbalance Piezoelectric Chemiluminescent Chemiluminescent Electrochemical

66.7–466.7 1.0–100.0 1.0–120 0.5–10 and 10.0–160

Incubation time (min)

DL (ng mL1)

Reference

30

0.4

[45]

60 35 10 35

1.2 0.22 0.14 1.5

[46] [38] [47] [48]

20 100 20 20

66.7 0.53 0.6 0.1

[49] [50] [51] This work

cretely, the proposed immunosensor was first incubated in 1 mL human serum samples for 20 min at 25 °C. Then, simply washed with bi-distilled water. Subsequently, the CV measurement was carried out in 0.02 M pH7.0 PBS. The test results were listed in Table 2. The results may imply that the detectable concentration of CEA in this assay satisfies the requirement of clinical analysis and that may provide a feasible alternative tool for the direct determination of CEA in real serum. 3.4.5. Characteristics comparison with other electrochemical CEA immunosensors The analytical performance of the developed CEA immunosensors has been compared with those of other CEA immunosensors reported in the literatures [51–57]. Characteristics such as linear ranges, detection limit (DL) and incubation time of every immunosensor were summarized in Table 3. As Table 3 shown, the linear ranges, DL, and incubation time of the proposed immunosensor are relative good. 4. Conclusions In the present paper, a novel approach for construction of an immunosensor was developed based on BSA/anti-CEA/nano-Au/ NiHCFNPs/nano-Au complex matrix modified electrode. The immunosensor can be used for directly monitoring the concentration of CEA in serum samples. The amperometric immunoassay strategy offered several advantages including simple fabrication, high sensitivity, low detection limit, satisfactory regenerations, quantitative immunobinding reactions, no need for labeled reagents, cost-effectiveness. Thus, this method could be further developed for practical clinical diagnosis of serum CEA level. Moreover, anti-CEA/nano-Au/NiHCFNPs/nano-Au efficiently immobilize antibody and could be used in the preparation of other amperometric immunosensors for detection of clinically or environmental interested biospecies. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 20675064), the Chinese Education Ministry

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