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Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels
Biochemical Engineering Journal 53 (2011) 223–228
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels Juan Tang a,1 , Jianxin Huang b,1 , Biling Su a , Huafeng Chen a , Dianping Tang a,∗ a b
Key Laboratory of Analysis and Detection for Food Safety (MOE and Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China Clinical Laboratory and Medical Diagnostics Laboratory, Fujian Provincial Hospital, Fuzhou 350001, PR China
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 21 October 2010 Accepted 7 November 2010
Keywords: Alpha-fetoprotein Carbon nanoparticles Conductivity Immunosensor Gold colloids Sandwich-type immunoassay
a b s t r a c t A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP-anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2 O2 –KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1–500 ng/mL with a detection limit of 50 pg/mL AFP at 3. The reproducibility and recovery were <10% and 83.9–112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Alpha-fetoprotein (AFP, ␣-fetoprotein), a major plasma protein produced by the yolk sac and the liver during fetal life, is thought to the fetal counterpart of serum albumin [1]. AFP is assayed in pregnant women, using maternal blood or amniotic fluid, as a screening test for a subset development abnormality. It is also measured in non-pregnant women, other adults, and children, serving as a biomarker to detect a subset of tumors [2]. In adults, levels over 500 ng/mL of AFP are seen in only three situations: hepatocellular carcinoma, germ cell tumors, and metastatic cancer in the liver originating from other primary tumors elsewhere [3]. According to the case loaded from our Medical Diagnostics Laboratory of Fujian Provincial Hospital in the recent two years, AFP and carcinoembryonic antigen (CEA) have become major detection indexes. Moreover, the number of serum specimens has been increasing, and there are almost more than 100 specimens for one day. Although the diagnostic methods are also continuously improved: from the early radioimmunoassay to the present Electrochemiluminescent Automatic Analyzer, it could not still meet the requirement of practical diagnosis to some extent. From our previous research for the
∗ Corresponding author. Tel.: +86 591 2286 6125; fax: +86 591 2286 6135. E-mail addresses: [email protected], [email protected] (D. Tang). 1 These authors contributed equally to this work. 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.11.001
electrochemical immunosensor [4–7], cheap, sensitive and rapid detection methods are preferable. Conductometric immunosensors have received great attention for the detection of tumor markers, since it is highly sensitive, low cost, low power requirements, and has high compatibility with advanced micromachining technologies without reference electrode and with low driving voltage, and suitable for sensor miniaturization and automated detection [8]. Simple conductivity sensors are constructed of an insulating material embedded with platinum, graphite, stainless steel or other metallic pieces [9]. These metal contacts serve as sensing elements and are placed at a fixed distance apart to make contact with a solution whose conductivity is to be determined [10]. The detection principle of conductometric enzyme immunoassay is based on the change in conductivity between two parallel electrodes through detecting the products of enzymatic reactions due to increasing conductivity of the enzyme membrane [11]. Liu et al. developed a micro-comb electrode-based conductometric immunosensor for the detection of aflatoxin B1 using nanogold particles as immobilized matrix, and the detection was based on the immobilized HRP as trace and H2 O2 , KI as enzyme substrates [12]. In addition, the conductometric immunoassay method was also employed for the detection of foodborne pathogens [13] and clinical diagnosis [14]. The advantages on conductometric sensor have been reviewed by several groups [15–17]. For the successful development of electrochemical immunoassay, signal amplification should be a critical issue [18,19]. Bioactive
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enzyme molecules are usually used as a labeling probe for the amplification of signal. However, the labeled amount of enzyme on each antibody is limited. The rapidly emerging research field of nanobiotechnology provides excitingly new possibilities for advanced development of new analytical tools and instrumentation for bioanalytical and biotechnological applications [20–22]. Liu et al. introduced a conductometric immunoassay for hepatitis B surface antigen using double-codified nanogold particles as labels, and the sensitivity could be greatly increased compared with the results obtained only using HRP-labeled hepatitis B surface antibody as detection antibodies [23]. Sergeyeva et al. reported a new conductometric sensor for detecting the presence and measuring the concentration of IgG in fluids using an electrically conducting polymer, polyaniline, as labels [24]. Carbon nanoparticles (CNPs) have been of considerable research attention due to their intrinsic chemical (sp2 – electrons) and physical (amorphous carbon nanoparticles) properties [25]. One major advantage using CNPs is that CNPs possessed unique properties including low density, high porosity and surface area, and relatively high chemical and thermal stability [26]. To the best of our knowledge, there is no report focusing on conductometric immunoassay of biomarkers using CNP-based nanolabels. Herein, we fabricated a sandwich-type conductometric immunoassay for the detection of AFP, as a model biomarker, using CNP-HRP-anti-AFP as detection antibodies for the amplification of conductometric signal. The aim of this study is to enhance the sensitivity of conventional sandwich-type conductometric immunoassay through changing the labeled method of detection antibodies. Relative experimental process and method were described as the following sections. 2. Experimental 2.1. Materials and reagents Monoclonal anti-fetoprotein primary antibody produced in mouse (anti-AFP, clone C3, immunogen: human ␣-fetoprotein) was purchased from Sigma–Aldrich. HRP-anti-AFP, and AFP standards were purchased from Biocell Biotechnol. Co. Ltd. (Zhengzhou, China). HAuCl4 ·4H2 O was purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide hydrochloride (EDC) were achieved from Sigma–Aldrich (St. Louis, MO). Carbon nanoparticles (CNPs; 14 nm in diameter; Printex 90) was obtained from Degussa (Frankfurt, Germany). The size, shape and characteristics of carbon nanoparticles have been described in detail in our recently published paper [26]. 0.08 M of phosphate-buffered saline (PBS, pH 7.4) was prepared by adding 12.2 g K2 HPO4 , 1.36 g KH2 PO4 , and 8.5 g NaCl into 1000 mL deionized water. All other reagents were of analytical grade and were used without further purification. Deionized and distilled water was used throughout the study. Hydrogen peroxide (H2 O2 ) standardized by iodimetric titration was freshly prepared in deionized water. 15 M of KI was prepared by dissolving in 0.08 M PBS (pH 7.4), immediately before use. Clinical serum samples were gifted from Fujian Provincial Hospital of China. 2.2. Apparatus and instruments The interdigitated transducers of the conductometric immunoassay were purchased from Institute of Chemo- and Biosensor (Chongqing, China). The transducer includes two identical pairs of gold interdigitated electrodes (150 nm thick) on the Pyrex glass substrate (10 mm × 30 mm). The sensitive part of each
Scheme 1. Fabrication process of CNP-HRP-anti-AFP, and measurement principle of conductometric immunoassay.
electrode was about 1 mm2 . Electrochemical measurements were carried out with an Electrochemical Quartz Crystal Microbalance (EQCM, CHI 430A, Shanghai CH Instruments Inc., China) using a conventional three-electrode system with a modified GCE as working electrode, a platinum foil as auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. Conductivity was measured using DDS-309+ Conductivity Meter (Chengdu, China). Ultraviolet–vis absorption (UV–vis) spectra were recorded with an 1102 UV–vis spectrophotometer (Techcomp, Shanghai, China). Clinical serum samples were assayed using Elecsys 2010 Electrochemiluminescent Automatic Analyzer (Roche, Switzerland). 2.3. Conjugation of HRP-anti-AFP with carbon nanoparticles 50 mg of carbon nanoparticles were initially treated with 10 mL of 3:1 H2 SO4 /HNO3 (v/v) in sonication for 4 h, and then the dispersion was centrifuged and washed repeatedly with water and ethanol at 10,000 rpm until pH was about 6.5. During this process, carboxylate groups were generated on the surface of CNPs. Following that, carbon nanoparticles were dissolved into 1 mL of pH 7.4 PBS. 13 mg of NHS and 18 mg of EDC were added into the solution followed with continuously stirring (750 rpm) for 45 min at room temperature (RT). Afterward, 300 L of HRP-anti-AFP (C[protein] = 53.4 mM) was dropped into the mixture, and stirred for 24 h at RT with a constant rate of 300 rpm. After completion of incubation, the CNP-HRP-anti-AFP conjugates (bionanolabels) were centrifuged for 20 min at 10,000 rpm. To block the residual active groups on the surface of CNPs, the bionanolabels were treated with 2.5 wt% BSA for 1 h at RT. Finally, the as-prepared bionanolabels were diluted into 5 mL of pH 7.4 PBS, and the concentration of CNP-HRP-anti-AFP was about 10 mg/mL. The conjugated process of CNP-HRP-anti-AFP is schematically illustrated in Scheme 1. 2.4. Fabrication of conductometric immunosensor Prior to experiment, the interdigitated electrode was slightly polished with 1.0 m alumina slurry, followed by successive sonication in bi-distilled water and ethanol for 5 min and dried in air. Gold nanoparticles were electrodeposited onto the surface of the electrode by immersing the clean electrode into the 1.0 wt% HAuCl4 solution with applying a constant potential of −0.2 V for 60 s. Following that, the nanogold-modified electrode was immersed into anti-AFP stock solution, and incubated for 12 h at 4 ◦ C to make anti-AFP assemble on the surface of nanogold particles. Afterward, the modified electrode was dipped into 1.0 wt% BSA for 60 min
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at room temperature to eliminate non-specific binding effect and block the remaining active groups. The resulting conductometric immunosensor was stored at 4 ◦ C when not in use. The fabrication procedure of the conductometric immunosensor is shown in Scheme 1. 2.5. Conductometric measurement The assayed process of the conductometric immunosensor toward the detection of AFP mainly consisted of three steps as follows: (i) the immunosensor was dipped into 50 L of AFP standard/sample with various concentrations, and incubated for 10 min at RT to form the antigen–antibody complex; (ii) the resulting immunosensor was immersed into 50 L of CNP-HRP-anti-AFP (10 mg/mL), and incubated for another 10 min at RT to form the sandwich-type immunocomplex; and (iii) the formed immunosensor was placed into 2 mL of pH 7.4 PBS containing 6 M H2 O2 and 15 M KI, and the conductivity (Sn , S) was recorded. All experiments were carried out at RT (25 ± 1.0 ◦ C). The calibration curve was plotted on the basis of the relationship between the conductivity and the concentration of AFP. Three replicate experiments were provided for all figures and tables. The detection principle is represented in Scheme 1.
Fig. 1. Cyclic voltammograms of the immunosensor after incubated with HRP-antiAFP-CNPs at the (a) absence and (b) presence of H2 O2 at 50 mV/s in pH 7.4 PBS.
278 nm for the HRP-anti-AFP. When the HRP-anti-AFP was conjugated onto the surface of carbon nanoparticles, the absorption peak was shifted to 263 nm. The reason might be attributed to the interaction between HRP-anti-AFP and carbon nanoparticles.
3. Results and discussion 3.2. Electrochemical characteristics of conductometric immunoassay
3.1. Construction and characteristics of conductometric immunoassay For the successful development of electrochemical immunoassay, the fabrication reproducibility of the immunosensor is critical. Various methods including encapsulation, absorption, covalent conjugation and self-assembly have been used the construction of electrochemical immunosensors. A simple and efficient immobilization method for biomolecules should be attractive. In this study, the primary anti-AFP antibodies were directly immobilized on the surface of electrodeposited nanogold particles using one-step strategy. The method could decrease the effect of artificial factors or environmental factors on the performance of the immunosensor during the fabricated process. Another key issue is to enhance the sensitivity of the immunoassay. Use of CNPs was expected to enhance the immobilized amount of biomolecules due to the physical and chemical properties of nanoparticles. With the sandwich-type immunoassay format, the antigen–antibody complex was formed via two-step incubation process as follows: anti-AFP + AFP → anti-AFP-AFP
(1)
anti-AFP-AFP + CNP-HRP-anti-AFP → anti-AFP-AFP-AFP-anti-HRP-CNP
(2)
The conductometric measurement was based on the change in the free iodine concentration in the detection solution due to bioelectrocatalytic reaction of the carried HRP in the sandwich immunocomplex: HRP(ox) + H2 O2 + 2I− + 2H+ → HRP(red) + 2H2 O + I2
(3)
Concluding, the formation of the sandwich-type immunocomplex introduced a local change of conductivity for the immunosensing interface. To further investigate the formation of bionanolabels, the absorption spectra of HRP-anti-AFP, CNPs and CNP-HRP-anti-AFP are characterized. A absorption peak at 250 nm was observed for the carboxylated CNPs, while there was one absorption peaks at
In the sandwich-type immunoassay, the signal mainly derived from the carried HRP. Thus, the bioactivity of HRP should be studied. To verify this issue, cyclic voltammograms of the sandwich immunocomplex were investigated using the bioelectrocatalytic reaction of the carried HRP toward H2 O2 . Fig. 1 shows the cyclic voltammograms of the sandwich immunocomplex-modified electrode in pH 7.4 PBS at the absence and presence of 6 M H2 O2 . Seen from Fig. 1, upon the addition of hydrogen peroxide into the substrate solution, an obvious catalytic characteristic was appeared with a dramatic increase of the reduction current and a sharp decrease of the oxidation current. This result indicated the carried HRP in the bionanolabels could retain high enzymatic catalytic activity, and effectively shuttle electrons from the base electrode surface to the redox center of HRP.
3.3. Verification of CNP-based amplified properties To verify the advantages of the CNP-based bionanolabels in the developed immunoassay, a comparative study was carried out using CNP-HRP-anti-AFP and HRP-anti-AFP as detection antibodies, respectively. Five levels of AFP including 1.0, 10, 50, 200 and 400 ng/mL, as examples, were assayed using the same-batch immunosensor with the same immunoassay format. As shown in Fig. 2, the immunoassay using CNP-HRP-anti-AFP as detection antibodies exhibited higher conductometric response than those of using HRP-anti-AFP as detection antibodies. The reason might be attributed to the following three points: (i) carbon nanoparticles with high surface-to-volume ratio and surface free energy could conjugate more HRP-anti-AFP molecules on the surface of one nanoparticle; (ii) using HRP-anti-AFP as detection antibodies, the rate between HRP-anti-AFP and AFP in the sandwich immunocomplex was small (almost 1:1); and (iii) using CNP-HRP-anti-AFP as detection antibodies, the other HRP-anti-AFP molecules could carry into the sandwich immunocomplex, and participate the bioelectrocatalytic reaction when one antibody on the surface of CNP reacted with AFP in the sample. Thus, the sensitivity could be improved and increased using CNP-HRP-anti-AFP as detection antibodies.
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Fig. 2. Conductometric responses of the immunosensor toward various AFP standards using (a) HRP-anti-AFP-CNPs and (b) HRP-anti-AFP as detection antibodies.
3.4. Optimization of external conditions During the formation of the conductometric immunosensor, the primary anti-AFP antibodies were immobilized on the surface of electrode via the electrodeposited nanogold particles. So, the surface coverage of gold nanoparticles on the electrode surface greatly affected the sensitivity of conductometric immunosensor. Fig. 3a shows the effect of electrodeposited time of gold nanoparticles on the sensitivity of the immunosensor (50 ng/mL AFP as an example). Seen from Fig. 3a, the conductometric responses decreased with the increment of electrodeposited time, and increased after 10 s. The reason might be the fact that a lot of nanogold particles were aggregated on the surface of the electrode after 10 s, and exhibited the properties of bulk gold. On the contrary, gold nanoparticles might be homogeneously disperse on the surface of the electrode, and displayed single-layer gold particles. So 10 s of electrodeposited time was used for the fabrication of the conductometric immunosensor. On the other hand, the amount of HRP-anti-AFP on the surface of CNPs would directly influence the sensitivity of the immunosensor. Various volumes of HRP-anti-AFP at the total protein concentration of 53.4 mM were used for the label of 50 mg CNPs. As shown in Fig. 3b, the conductometric responses decreased with the volume of HRP-anti-AFP increased, and leveled off at 300 L. The reason was attributed to the saturation of HRP-anti-AFP on the nanoparticle surface. Thus, 300 L HRP-anti-AFP (C[protein] = 53.4 mM) was employed for the label of 50 mg CNPs. In general, the immunocomplex could be increased with the increment of incubation temperature. However, the temperature greatly influenced the conductometric response of the immunosensor. Thus, all experiments were carried out at room temperature (25 ± 1.0 ◦ C). With this condition, the effect of various incubation
Fig. 4. Calibration curve of the conductometric immunosensor toward AFP standards in 0.08 M pH 7.4 PBS containing 6 M H2 O2 and 15 M KI (Inset: linear curve).
times on the conductivity of the immunosensor was investigated. Seen from Fig. 3c, the conductivity increased with the increasing incubation time, and trended to level off after 10 min. Longer incubation time did not improve the response. So, 10 min was used for the antigen–antibody reaction. 3.5. Analytical performance To evaluate the sensitivity and dynamic range of the conductometric immunosensor, a sandwich-type assay format was applied for the detection of AFP using CNP-HRP-anti-AFP as detection antibodies. A sigmoid curve regression between the conductivity and the concentration of AFP was obtained (Fig. 4). The conductivity decreased with the increase of the AFP concentration. As shown in the inset of Fig. 4, the decrease of conductivity was linear in the range of 0.1 to 500 ng/mL and the linear regression equation was adjusted to S (S) = 27.57 − 4.65 × Log C[AFP] (ng/mL) with a detection limit (LOD) of 50 pg/mL at a signal to noise ratio of 3 (where is the standard deviation of the blank, n = 15) (R2 = 0.981). Since the cutoff value of serum AFP in diagnostic is 10 ng/mL, the sensitivity of the immunosensor was enough to practical application. Moreover, the LOD was obviously lower than that of commercially available enzymelinked immunosorbent assay (ELISA with a LOD of 1.0 ng/mL AFP, http://www.caigou.com.cn/netshow/E00054000/CP/241653.html). Significantly, the system was capable of continuously carrying out all steps in less than 25 min for one sample, which is shorter than that of the commercial ELISA (>4 h). The precision of the conductometric immunoassay was evaluated by calculating the intra- and inter-assay variation coefficients (CVs, n = 5). Experimental results indicated that the CVs of the intra-
Fig. 3. Effect of (a) electrodeposited time of gold nanoparticles, (b) HRP-anti-AFP (C[protein] = 53.4 mM), and (c) incubation time on the conductometric responses of the immunosensor (Note: 50 ng/mL AFP as an example).
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Table 1 Interference degree or crossing recognition level of the conductometric immunosensor. Crossing reagentsa
AFP + CEA AFP + CA 125 AFP + CA 19-9 AFP + BSA a b
Mean ± SD (S)
C[interfering agents] (ng/mL or U/mL); conductivity (S)b 0
5
50
100
200
6.7 6.8 6.7 6.8
7.1 7.3 6.2 6.5
8.5 7.9 6.9 6.6
8.3 8.4 7.4 7.1
8.2 8.5 8.1 7.4
7.76 7.78 7.06 6.88
± ± ± ±
0.72 0.65 0.65 0.33
RSD (%)
9.3 8.4 9.2 4.8
Containing 50 ng/mL AFP and interfering agents with various concentrations. The average value of three successful assays.
Table 2 The recoveries assayed by using the conductometric immunosensor via spiking AFP standards into blank cattle serum. Sample no.
Spiking value (ng/mL)
Assayed value (mean ± SD, ng/mL)a
1 2 3 4 5 6
5.6 165.8 56.5 269.2 34.9 89.7
4.7 176.4 48.2 234.1 39.2 78.7
a
± ± ± ± ± ±
0.6 5.7 3.1 13.4 4.3 3.9
Recovery (%) 83.9 106.4 85.3 87.0 112.3 87.7
The average of three successful assays.
assay were 8.4%, 6.9%, and 9.3% at 1.0, 50, and 300 ng/mL AFP, respectively, while the CVs of the inter-assay using nanoparticles from different batches were 9.6%, 8.7%, and 9.2% at the abovementioned analyte concentrations. The bionanolabels exhibited satisfactory stability. In fact, 89.7% of the initial conductivity was obtained in the immunoassay after storage of the particles at 4 ◦ C for 27 days. Further, the effects of potentially interfering components on the detection of AFP in serum sample were evaluated, such as carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), and BSA. Conductometric responses of the immunosensor in 0.5, 50, 200, and 400 ng/mL AFP standards containing interfering substances of different concentrations were assayed, and the RSD values were 4.3–9.7%, 4.8–9.3%, 3.7–10.4%, and 4.3–8.6% for 0.5, 50, 200, and 400 ng/mL AFP, respectively. Table 1 shows the experimental data in 50 ng/mL (as an example) of AFP solutions containing various interfering substrates. So the selectivity of the as-prepared immunosensor was acceptable.
Fig. 5. Comparison of assayed results using the conductometric immunosensor and ECL referenced method.
4. Conclusion This contribution describes a new sandwich-type conductometric immunosensor for the detection of AFP using carbon nanoparticles-functionalized biomolecules as detection antibodies. Compared with conventional sandwich-type immunoassay, the presence of carbon nanoparticles could obviously improve the sensitivity of the conductometric immunosensor. Meanwhile, HRP-anti-AFP-conjugated carbon nanoparticles exhibited good dispersion in the solution. The highlight of this work is to fabricate a conductive and non-toxic bionanolabel for the signal amplification of the conductometric immunoassay. This provides a promising and facile pathway for the sandwich-type conductometric immunoassay. Acknowledgements
3.6. Analysis of human serum samples and method comparison Six AFP standards with various concentrations were spiked into the blank cattle serum, and these samples were assayed using the developed conductometric immunosensor. As indicated in Table 2, the recoveries of two samples were above 100% while those of four samples were below 100%. The reason for the above 100% might be the fact that the binding antigens were not completely released from the electrode during the regeneration, and the remained antigen indirectly increased the binding amount of HRP-anti-AFP. Considering this issue, the recovery of the proposed immunosensor was acceptable. To further investigate the possibility of the proposed immunoassay for real samples, 45 serum specimens were evaluated by the developed immunoassay and the Electrochemiluminescent (ECL) method (Roche 2010 Electrochemiluminescent Automatic Analyzer). The regression equation (linear) for these data is as follows (x-axis, ECL; y-axis, immunosensor): y = 0.969(±0.234)x − 0.591(±4.67) (R2 = 0.988) for AFP (Fig. 5). These data show no significant difference between the two methods.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels Juan Tang a,1 , Jianxin Huang b,1 , Biling Su a , Huafeng Chen a , Dianping Tang a,∗ a b
Key Laboratory of Analysis and Detection for Food Safety (MOE and Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China Clinical Laboratory and Medical Diagnostics Laboratory, Fujian Provincial Hospital, Fuzhou 350001, PR China
a r t i c l e
i n f o
Article history: Received 8 July 2010 Received in revised form 21 October 2010 Accepted 7 November 2010
Keywords: Alpha-fetoprotein Carbon nanoparticles Conductivity Immunosensor Gold colloids Sandwich-type immunoassay
a b s t r a c t A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP-anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2 O2 –KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1–500 ng/mL with a detection limit of 50 pg/mL AFP at 3. The reproducibility and recovery were <10% and 83.9–112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Alpha-fetoprotein (AFP, ␣-fetoprotein), a major plasma protein produced by the yolk sac and the liver during fetal life, is thought to the fetal counterpart of serum albumin [1]. AFP is assayed in pregnant women, using maternal blood or amniotic fluid, as a screening test for a subset development abnormality. It is also measured in non-pregnant women, other adults, and children, serving as a biomarker to detect a subset of tumors [2]. In adults, levels over 500 ng/mL of AFP are seen in only three situations: hepatocellular carcinoma, germ cell tumors, and metastatic cancer in the liver originating from other primary tumors elsewhere [3]. According to the case loaded from our Medical Diagnostics Laboratory of Fujian Provincial Hospital in the recent two years, AFP and carcinoembryonic antigen (CEA) have become major detection indexes. Moreover, the number of serum specimens has been increasing, and there are almost more than 100 specimens for one day. Although the diagnostic methods are also continuously improved: from the early radioimmunoassay to the present Electrochemiluminescent Automatic Analyzer, it could not still meet the requirement of practical diagnosis to some extent. From our previous research for the
∗ Corresponding author. Tel.: +86 591 2286 6125; fax: +86 591 2286 6135. E-mail addresses: [email protected], [email protected] (D. Tang). 1 These authors contributed equally to this work. 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.11.001
electrochemical immunosensor [4–7], cheap, sensitive and rapid detection methods are preferable. Conductometric immunosensors have received great attention for the detection of tumor markers, since it is highly sensitive, low cost, low power requirements, and has high compatibility with advanced micromachining technologies without reference electrode and with low driving voltage, and suitable for sensor miniaturization and automated detection [8]. Simple conductivity sensors are constructed of an insulating material embedded with platinum, graphite, stainless steel or other metallic pieces [9]. These metal contacts serve as sensing elements and are placed at a fixed distance apart to make contact with a solution whose conductivity is to be determined [10]. The detection principle of conductometric enzyme immunoassay is based on the change in conductivity between two parallel electrodes through detecting the products of enzymatic reactions due to increasing conductivity of the enzyme membrane [11]. Liu et al. developed a micro-comb electrode-based conductometric immunosensor for the detection of aflatoxin B1 using nanogold particles as immobilized matrix, and the detection was based on the immobilized HRP as trace and H2 O2 , KI as enzyme substrates [12]. In addition, the conductometric immunoassay method was also employed for the detection of foodborne pathogens [13] and clinical diagnosis [14]. The advantages on conductometric sensor have been reviewed by several groups [15–17]. For the successful development of electrochemical immunoassay, signal amplification should be a critical issue [18,19]. Bioactive
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enzyme molecules are usually used as a labeling probe for the amplification of signal. However, the labeled amount of enzyme on each antibody is limited. The rapidly emerging research field of nanobiotechnology provides excitingly new possibilities for advanced development of new analytical tools and instrumentation for bioanalytical and biotechnological applications [20–22]. Liu et al. introduced a conductometric immunoassay for hepatitis B surface antigen using double-codified nanogold particles as labels, and the sensitivity could be greatly increased compared with the results obtained only using HRP-labeled hepatitis B surface antibody as detection antibodies [23]. Sergeyeva et al. reported a new conductometric sensor for detecting the presence and measuring the concentration of IgG in fluids using an electrically conducting polymer, polyaniline, as labels [24]. Carbon nanoparticles (CNPs) have been of considerable research attention due to their intrinsic chemical (sp2 – electrons) and physical (amorphous carbon nanoparticles) properties [25]. One major advantage using CNPs is that CNPs possessed unique properties including low density, high porosity and surface area, and relatively high chemical and thermal stability [26]. To the best of our knowledge, there is no report focusing on conductometric immunoassay of biomarkers using CNP-based nanolabels. Herein, we fabricated a sandwich-type conductometric immunoassay for the detection of AFP, as a model biomarker, using CNP-HRP-anti-AFP as detection antibodies for the amplification of conductometric signal. The aim of this study is to enhance the sensitivity of conventional sandwich-type conductometric immunoassay through changing the labeled method of detection antibodies. Relative experimental process and method were described as the following sections. 2. Experimental 2.1. Materials and reagents Monoclonal anti-fetoprotein primary antibody produced in mouse (anti-AFP, clone C3, immunogen: human ␣-fetoprotein) was purchased from Sigma–Aldrich. HRP-anti-AFP, and AFP standards were purchased from Biocell Biotechnol. Co. Ltd. (Zhengzhou, China). HAuCl4 ·4H2 O was purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide hydrochloride (EDC) were achieved from Sigma–Aldrich (St. Louis, MO). Carbon nanoparticles (CNPs; 14 nm in diameter; Printex 90) was obtained from Degussa (Frankfurt, Germany). The size, shape and characteristics of carbon nanoparticles have been described in detail in our recently published paper [26]. 0.08 M of phosphate-buffered saline (PBS, pH 7.4) was prepared by adding 12.2 g K2 HPO4 , 1.36 g KH2 PO4 , and 8.5 g NaCl into 1000 mL deionized water. All other reagents were of analytical grade and were used without further purification. Deionized and distilled water was used throughout the study. Hydrogen peroxide (H2 O2 ) standardized by iodimetric titration was freshly prepared in deionized water. 15 M of KI was prepared by dissolving in 0.08 M PBS (pH 7.4), immediately before use. Clinical serum samples were gifted from Fujian Provincial Hospital of China. 2.2. Apparatus and instruments The interdigitated transducers of the conductometric immunoassay were purchased from Institute of Chemo- and Biosensor (Chongqing, China). The transducer includes two identical pairs of gold interdigitated electrodes (150 nm thick) on the Pyrex glass substrate (10 mm × 30 mm). The sensitive part of each
Scheme 1. Fabrication process of CNP-HRP-anti-AFP, and measurement principle of conductometric immunoassay.
electrode was about 1 mm2 . Electrochemical measurements were carried out with an Electrochemical Quartz Crystal Microbalance (EQCM, CHI 430A, Shanghai CH Instruments Inc., China) using a conventional three-electrode system with a modified GCE as working electrode, a platinum foil as auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. Conductivity was measured using DDS-309+ Conductivity Meter (Chengdu, China). Ultraviolet–vis absorption (UV–vis) spectra were recorded with an 1102 UV–vis spectrophotometer (Techcomp, Shanghai, China). Clinical serum samples were assayed using Elecsys 2010 Electrochemiluminescent Automatic Analyzer (Roche, Switzerland). 2.3. Conjugation of HRP-anti-AFP with carbon nanoparticles 50 mg of carbon nanoparticles were initially treated with 10 mL of 3:1 H2 SO4 /HNO3 (v/v) in sonication for 4 h, and then the dispersion was centrifuged and washed repeatedly with water and ethanol at 10,000 rpm until pH was about 6.5. During this process, carboxylate groups were generated on the surface of CNPs. Following that, carbon nanoparticles were dissolved into 1 mL of pH 7.4 PBS. 13 mg of NHS and 18 mg of EDC were added into the solution followed with continuously stirring (750 rpm) for 45 min at room temperature (RT). Afterward, 300 L of HRP-anti-AFP (C[protein] = 53.4 mM) was dropped into the mixture, and stirred for 24 h at RT with a constant rate of 300 rpm. After completion of incubation, the CNP-HRP-anti-AFP conjugates (bionanolabels) were centrifuged for 20 min at 10,000 rpm. To block the residual active groups on the surface of CNPs, the bionanolabels were treated with 2.5 wt% BSA for 1 h at RT. Finally, the as-prepared bionanolabels were diluted into 5 mL of pH 7.4 PBS, and the concentration of CNP-HRP-anti-AFP was about 10 mg/mL. The conjugated process of CNP-HRP-anti-AFP is schematically illustrated in Scheme 1. 2.4. Fabrication of conductometric immunosensor Prior to experiment, the interdigitated electrode was slightly polished with 1.0 m alumina slurry, followed by successive sonication in bi-distilled water and ethanol for 5 min and dried in air. Gold nanoparticles were electrodeposited onto the surface of the electrode by immersing the clean electrode into the 1.0 wt% HAuCl4 solution with applying a constant potential of −0.2 V for 60 s. Following that, the nanogold-modified electrode was immersed into anti-AFP stock solution, and incubated for 12 h at 4 ◦ C to make anti-AFP assemble on the surface of nanogold particles. Afterward, the modified electrode was dipped into 1.0 wt% BSA for 60 min
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at room temperature to eliminate non-specific binding effect and block the remaining active groups. The resulting conductometric immunosensor was stored at 4 ◦ C when not in use. The fabrication procedure of the conductometric immunosensor is shown in Scheme 1. 2.5. Conductometric measurement The assayed process of the conductometric immunosensor toward the detection of AFP mainly consisted of three steps as follows: (i) the immunosensor was dipped into 50 L of AFP standard/sample with various concentrations, and incubated for 10 min at RT to form the antigen–antibody complex; (ii) the resulting immunosensor was immersed into 50 L of CNP-HRP-anti-AFP (10 mg/mL), and incubated for another 10 min at RT to form the sandwich-type immunocomplex; and (iii) the formed immunosensor was placed into 2 mL of pH 7.4 PBS containing 6 M H2 O2 and 15 M KI, and the conductivity (Sn , S) was recorded. All experiments were carried out at RT (25 ± 1.0 ◦ C). The calibration curve was plotted on the basis of the relationship between the conductivity and the concentration of AFP. Three replicate experiments were provided for all figures and tables. The detection principle is represented in Scheme 1.
Fig. 1. Cyclic voltammograms of the immunosensor after incubated with HRP-antiAFP-CNPs at the (a) absence and (b) presence of H2 O2 at 50 mV/s in pH 7.4 PBS.
278 nm for the HRP-anti-AFP. When the HRP-anti-AFP was conjugated onto the surface of carbon nanoparticles, the absorption peak was shifted to 263 nm. The reason might be attributed to the interaction between HRP-anti-AFP and carbon nanoparticles.
3. Results and discussion 3.2. Electrochemical characteristics of conductometric immunoassay
3.1. Construction and characteristics of conductometric immunoassay For the successful development of electrochemical immunoassay, the fabrication reproducibility of the immunosensor is critical. Various methods including encapsulation, absorption, covalent conjugation and self-assembly have been used the construction of electrochemical immunosensors. A simple and efficient immobilization method for biomolecules should be attractive. In this study, the primary anti-AFP antibodies were directly immobilized on the surface of electrodeposited nanogold particles using one-step strategy. The method could decrease the effect of artificial factors or environmental factors on the performance of the immunosensor during the fabricated process. Another key issue is to enhance the sensitivity of the immunoassay. Use of CNPs was expected to enhance the immobilized amount of biomolecules due to the physical and chemical properties of nanoparticles. With the sandwich-type immunoassay format, the antigen–antibody complex was formed via two-step incubation process as follows: anti-AFP + AFP → anti-AFP-AFP
(1)
anti-AFP-AFP + CNP-HRP-anti-AFP → anti-AFP-AFP-AFP-anti-HRP-CNP
(2)
The conductometric measurement was based on the change in the free iodine concentration in the detection solution due to bioelectrocatalytic reaction of the carried HRP in the sandwich immunocomplex: HRP(ox) + H2 O2 + 2I− + 2H+ → HRP(red) + 2H2 O + I2
(3)
Concluding, the formation of the sandwich-type immunocomplex introduced a local change of conductivity for the immunosensing interface. To further investigate the formation of bionanolabels, the absorption spectra of HRP-anti-AFP, CNPs and CNP-HRP-anti-AFP are characterized. A absorption peak at 250 nm was observed for the carboxylated CNPs, while there was one absorption peaks at
In the sandwich-type immunoassay, the signal mainly derived from the carried HRP. Thus, the bioactivity of HRP should be studied. To verify this issue, cyclic voltammograms of the sandwich immunocomplex were investigated using the bioelectrocatalytic reaction of the carried HRP toward H2 O2 . Fig. 1 shows the cyclic voltammograms of the sandwich immunocomplex-modified electrode in pH 7.4 PBS at the absence and presence of 6 M H2 O2 . Seen from Fig. 1, upon the addition of hydrogen peroxide into the substrate solution, an obvious catalytic characteristic was appeared with a dramatic increase of the reduction current and a sharp decrease of the oxidation current. This result indicated the carried HRP in the bionanolabels could retain high enzymatic catalytic activity, and effectively shuttle electrons from the base electrode surface to the redox center of HRP.
3.3. Verification of CNP-based amplified properties To verify the advantages of the CNP-based bionanolabels in the developed immunoassay, a comparative study was carried out using CNP-HRP-anti-AFP and HRP-anti-AFP as detection antibodies, respectively. Five levels of AFP including 1.0, 10, 50, 200 and 400 ng/mL, as examples, were assayed using the same-batch immunosensor with the same immunoassay format. As shown in Fig. 2, the immunoassay using CNP-HRP-anti-AFP as detection antibodies exhibited higher conductometric response than those of using HRP-anti-AFP as detection antibodies. The reason might be attributed to the following three points: (i) carbon nanoparticles with high surface-to-volume ratio and surface free energy could conjugate more HRP-anti-AFP molecules on the surface of one nanoparticle; (ii) using HRP-anti-AFP as detection antibodies, the rate between HRP-anti-AFP and AFP in the sandwich immunocomplex was small (almost 1:1); and (iii) using CNP-HRP-anti-AFP as detection antibodies, the other HRP-anti-AFP molecules could carry into the sandwich immunocomplex, and participate the bioelectrocatalytic reaction when one antibody on the surface of CNP reacted with AFP in the sample. Thus, the sensitivity could be improved and increased using CNP-HRP-anti-AFP as detection antibodies.
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Fig. 2. Conductometric responses of the immunosensor toward various AFP standards using (a) HRP-anti-AFP-CNPs and (b) HRP-anti-AFP as detection antibodies.
3.4. Optimization of external conditions During the formation of the conductometric immunosensor, the primary anti-AFP antibodies were immobilized on the surface of electrode via the electrodeposited nanogold particles. So, the surface coverage of gold nanoparticles on the electrode surface greatly affected the sensitivity of conductometric immunosensor. Fig. 3a shows the effect of electrodeposited time of gold nanoparticles on the sensitivity of the immunosensor (50 ng/mL AFP as an example). Seen from Fig. 3a, the conductometric responses decreased with the increment of electrodeposited time, and increased after 10 s. The reason might be the fact that a lot of nanogold particles were aggregated on the surface of the electrode after 10 s, and exhibited the properties of bulk gold. On the contrary, gold nanoparticles might be homogeneously disperse on the surface of the electrode, and displayed single-layer gold particles. So 10 s of electrodeposited time was used for the fabrication of the conductometric immunosensor. On the other hand, the amount of HRP-anti-AFP on the surface of CNPs would directly influence the sensitivity of the immunosensor. Various volumes of HRP-anti-AFP at the total protein concentration of 53.4 mM were used for the label of 50 mg CNPs. As shown in Fig. 3b, the conductometric responses decreased with the volume of HRP-anti-AFP increased, and leveled off at 300 L. The reason was attributed to the saturation of HRP-anti-AFP on the nanoparticle surface. Thus, 300 L HRP-anti-AFP (C[protein] = 53.4 mM) was employed for the label of 50 mg CNPs. In general, the immunocomplex could be increased with the increment of incubation temperature. However, the temperature greatly influenced the conductometric response of the immunosensor. Thus, all experiments were carried out at room temperature (25 ± 1.0 ◦ C). With this condition, the effect of various incubation
Fig. 4. Calibration curve of the conductometric immunosensor toward AFP standards in 0.08 M pH 7.4 PBS containing 6 M H2 O2 and 15 M KI (Inset: linear curve).
times on the conductivity of the immunosensor was investigated. Seen from Fig. 3c, the conductivity increased with the increasing incubation time, and trended to level off after 10 min. Longer incubation time did not improve the response. So, 10 min was used for the antigen–antibody reaction. 3.5. Analytical performance To evaluate the sensitivity and dynamic range of the conductometric immunosensor, a sandwich-type assay format was applied for the detection of AFP using CNP-HRP-anti-AFP as detection antibodies. A sigmoid curve regression between the conductivity and the concentration of AFP was obtained (Fig. 4). The conductivity decreased with the increase of the AFP concentration. As shown in the inset of Fig. 4, the decrease of conductivity was linear in the range of 0.1 to 500 ng/mL and the linear regression equation was adjusted to S (S) = 27.57 − 4.65 × Log C[AFP] (ng/mL) with a detection limit (LOD) of 50 pg/mL at a signal to noise ratio of 3 (where is the standard deviation of the blank, n = 15) (R2 = 0.981). Since the cutoff value of serum AFP in diagnostic is 10 ng/mL, the sensitivity of the immunosensor was enough to practical application. Moreover, the LOD was obviously lower than that of commercially available enzymelinked immunosorbent assay (ELISA with a LOD of 1.0 ng/mL AFP, http://www.caigou.com.cn/netshow/E00054000/CP/241653.html). Significantly, the system was capable of continuously carrying out all steps in less than 25 min for one sample, which is shorter than that of the commercial ELISA (>4 h). The precision of the conductometric immunoassay was evaluated by calculating the intra- and inter-assay variation coefficients (CVs, n = 5). Experimental results indicated that the CVs of the intra-
Fig. 3. Effect of (a) electrodeposited time of gold nanoparticles, (b) HRP-anti-AFP (C[protein] = 53.4 mM), and (c) incubation time on the conductometric responses of the immunosensor (Note: 50 ng/mL AFP as an example).
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Table 1 Interference degree or crossing recognition level of the conductometric immunosensor. Crossing reagentsa
AFP + CEA AFP + CA 125 AFP + CA 19-9 AFP + BSA a b
Mean ± SD (S)
C[interfering agents] (ng/mL or U/mL); conductivity (S)b 0
5
50
100
200
6.7 6.8 6.7 6.8
7.1 7.3 6.2 6.5
8.5 7.9 6.9 6.6
8.3 8.4 7.4 7.1
8.2 8.5 8.1 7.4
7.76 7.78 7.06 6.88
± ± ± ±
0.72 0.65 0.65 0.33
RSD (%)
9.3 8.4 9.2 4.8
Containing 50 ng/mL AFP and interfering agents with various concentrations. The average value of three successful assays.
Table 2 The recoveries assayed by using the conductometric immunosensor via spiking AFP standards into blank cattle serum. Sample no.
Spiking value (ng/mL)
Assayed value (mean ± SD, ng/mL)a
1 2 3 4 5 6
5.6 165.8 56.5 269.2 34.9 89.7
4.7 176.4 48.2 234.1 39.2 78.7
a
± ± ± ± ± ±
0.6 5.7 3.1 13.4 4.3 3.9
Recovery (%) 83.9 106.4 85.3 87.0 112.3 87.7
The average of three successful assays.
assay were 8.4%, 6.9%, and 9.3% at 1.0, 50, and 300 ng/mL AFP, respectively, while the CVs of the inter-assay using nanoparticles from different batches were 9.6%, 8.7%, and 9.2% at the abovementioned analyte concentrations. The bionanolabels exhibited satisfactory stability. In fact, 89.7% of the initial conductivity was obtained in the immunoassay after storage of the particles at 4 ◦ C for 27 days. Further, the effects of potentially interfering components on the detection of AFP in serum sample were evaluated, such as carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), and BSA. Conductometric responses of the immunosensor in 0.5, 50, 200, and 400 ng/mL AFP standards containing interfering substances of different concentrations were assayed, and the RSD values were 4.3–9.7%, 4.8–9.3%, 3.7–10.4%, and 4.3–8.6% for 0.5, 50, 200, and 400 ng/mL AFP, respectively. Table 1 shows the experimental data in 50 ng/mL (as an example) of AFP solutions containing various interfering substrates. So the selectivity of the as-prepared immunosensor was acceptable.
Fig. 5. Comparison of assayed results using the conductometric immunosensor and ECL referenced method.
4. Conclusion This contribution describes a new sandwich-type conductometric immunosensor for the detection of AFP using carbon nanoparticles-functionalized biomolecules as detection antibodies. Compared with conventional sandwich-type immunoassay, the presence of carbon nanoparticles could obviously improve the sensitivity of the conductometric immunosensor. Meanwhile, HRP-anti-AFP-conjugated carbon nanoparticles exhibited good dispersion in the solution. The highlight of this work is to fabricate a conductive and non-toxic bionanolabel for the signal amplification of the conductometric immunoassay. This provides a promising and facile pathway for the sandwich-type conductometric immunoassay. Acknowledgements
3.6. Analysis of human serum samples and method comparison Six AFP standards with various concentrations were spiked into the blank cattle serum, and these samples were assayed using the developed conductometric immunosensor. As indicated in Table 2, the recoveries of two samples were above 100% while those of four samples were below 100%. The reason for the above 100% might be the fact that the binding antigens were not completely released from the electrode during the regeneration, and the remained antigen indirectly increased the binding amount of HRP-anti-AFP. Considering this issue, the recovery of the proposed immunosensor was acceptable. To further investigate the possibility of the proposed immunoassay for real samples, 45 serum specimens were evaluated by the developed immunoassay and the Electrochemiluminescent (ECL) method (Roche 2010 Electrochemiluminescent Automatic Analyzer). The regression equation (linear) for these data is as follows (x-axis, ECL; y-axis, immunosensor): y = 0.969(±0.234)x − 0.591(±4.67) (R2 = 0.988) for AFP (Fig. 5). These data show no significant difference between the two methods.
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