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Enzyme–antibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein
Biologicals xxx (2017) 1e6
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Biologicals journal homepage: www.elsevier.com/locate/biologicals
Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein Qian Xiao, Yizhi Zheng, Jing Liu, Suiping Wang, Bo Feng* College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2016 Received in revised form 20 February 2017 Accepted 28 February 2017 Available online xxx
In this study, we designed a comprehensive strategy for the ultrasensitive detection of alpha fetoprotein (AFP) with high specificity using gold nanoparticle (AuNP)-based enzyme-linked immunosorbent assay (ELISA). A dual-film modified probe was synthesized by coating AuNPs with horseradish peroxidase (HRP) on its surface. Anti-AFP monoclonal antibody (McAb) was immobilized on the surface of the enzyme using glutaraldehyde cross-linking method. AuNPs, employed as support for the immobilization of HRP. HRP was used not only as the enzymatic-amplified tracer but also as a bridge for loading McAb. The limit of detection was 2 ng mL-1. The developed probes can provide an alternative approach with high sensitivity and a simple process similar to that of the traditional HRP-McAb based ELISA for the ultrasensitive detection of AFP in serum. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Gold nanoparticle Nanoprobe AFP ELISA
1. Introduction Serum alpha fetoprotein (AFP) has been extensively used as a biomarker for liver cancer, which is the second leading cause of cancer death in male worldwide [1]. Additionally, hepatocellular carcinoma (HCC) surveillance using ultrasound combined with AFP measurement is widely applied in practice. Strategies that enhance the sensitivity of existing surveillance tests can improve the feasibility of the early detection of HCC and may provide a more effective method for HCC surveillance [2]. Therefore, a sensitive detection and an accurate analysis of AFP concentrations are important. Various analytical techniques, such as electrochemiluminescence immunoassay [3], radioimmunoassay [4], imaging ellipsometry [5], and localized surface plasmon-coupled fluorescence [6] have been developed to determine AFP in human serum. These techniques are sensitive and highly specific for AFP detection. However, these methods are unsuitable for a low-cost, convenient, and rapid determination of AFP because they require skilled analysts and delicate instruments. ELISA, which is based on antibodyeantigen immunoreactions, is a powerful method in biological studies because of its convenient
* Corresponding author. Biological Engineering Department, College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China. E-mail address: [email protected] (B. Feng).
operation, capability of simultaneous measurement of large sample size, and low cost [7]. This approach is also the main method for measuring AFP. However, higher sensitivity is required for a precise diagnosis in clinical applications. Colloidal gold nanoparticles (AuNPs) have earned increasing attention in recent years in bioanalytical application including clinical diagnostics, point-of-care testing and therapeutic research as well as food safety and environmental monitoring [8]. One major merit of using AuNPs is that one can control and tailor their properties in a very predictable manner to meet the needs of specific application [9,10]. Therefore, AuNPs are extensively used for signal amplification in bioanalytical applications [11]. There are two main trends were well-established, one is utilizing the intrinsic properties of AuNPs such as color [12], localized surface plasmons [13], fluorescence resonance energy transfer [14], and many others. Another is carrying numerous biomolecules (e.g. antibody and enzyme molecule) and therefore generated a significant increase in signal [15]. Gao and co-workers designed a new ELISA strategy for highly sensitive colorimetric detection of prostate-specific antigen (PSA) by using AuNPs as the carriers of palladium nanostructures [16]. Li et al. synthesized a dual labeled probe by coating AuNPs with McAb and HRP on their surface. Based on the probe, an immunoassay was ultrasensitive detection of k-casein in bovine milk samples [17]. The same amplification technique also has been demonstrated in immunomagnetic beads separation [18]. In this study, AuNPs were employed to synthesize an ultrasensitive probe by dual-film assembly with HRP and anti-AFP
http://dx.doi.org/10.1016/j.biologicals.2017.02.008 1045-1056/© 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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Q. Xiao et al. / Biologicals xxx (2017) 1e6
monoclonal antibody (McAb) on the AuNP surface. The introduction of dual-film modified AuNPs into ELISA resulted in an ultrasensitive signal amplification to quantitatively analyze AFP. 2. Materials and methods 2.1. Chemicals and reagents Chloroauric acid (HAuCl4) was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Polystyrene 96-well microtiter plates were obtained from Jet Bio-Filtration Products, Co., Ltd. (China). Mouse anti-AFP McAb was procured from Aoke Biotech Co., Ltd. (China). Rabbit anti-AFP polyclonal antibody (PcAb) was procured from Wuhan Huamei Biotech Co., Ltd. (China). HRPconjugated mouse anti-AFP antibody was obtained from SigmaAldrich (USA). AFP from human fetal cord serum and HRP were purchased from Sino Biological Inc. (China). 3,30 ,5,50 -tetramethylbenzidine (TMB), H2O2, and Tween-20 were supplied by Shanghai Macklin Biochemical Co., Ltd. (China). Alkaline phosphatase (ALP), osteopontin (OPN) and ovalbumin (OVA) were procured from Yeasen Biological Technology Co., Ltd. (China). The various sera were obtained from CellMax Co., Ltd. (China). All other chemicals were of analytical grade and obtained from Aladdin Co., Ltd. (China). 2.2. Solutions and buffers Phosphate-buffered saline (PBS, pH 7.4) containing 137 mmol LNaCl, 2.7 mmol L-1 KCl, 10 mmol L-1 Na2HPO4, and 2 mmol L1 KH2PO4. PBS solution with 0.05% Tween-20 (v/v) (PBST) was used as washing buffer. Coating buffer was 50 mmol L-1 carbonate buffer (pH 9.5). TMB solution consisted of 50 mmol L-1 sodium citrate buffer (pH 5.0) containing 0.01% (w/v) TMB and 0.005% (v/v) H2O2. The stop solution was 2 mol L-1 H2SO4. Deionized water (18 MU cm at 25 C) was used throughout the experiments. 1
2.3. Preparations of AuNPs AuNPs were prepared according to the chemical reduction method by Frens [19] with slight modification. All glassware was thoroughly cleaned in aqua regia [3v HCl/1v HNO3], rinsed in deionized water, and oven-dried prior to use. Then, 2 mL of 1% HAuCl4 solution in 200 mL of deionized water was boiled thoroughly with continuous stirring, and 4 mL of 1% trisodium citrate solution was quickly added under vigorous stirring. The solution color changed from gray to purple to wine-red. The solution was boiled for another 10 min. The heating source was removed, and the solution was continuously stirred for another 15 min. The solution was cooled to room temperature and stored in a dark bottle at 4 C. 2.4. Dual-film modified AuNPs The pH of AuNP solution was adjusted to 8.6 with 0.1 M Na2CO3. Then, 200 mL of 1 mg/mL HRP solution was added into 10 mL of the AuNP solution under agitation for 20 min. The solution was allowed to stand at 25 C without mixing for 2 h. Then, 100 mL of 1% (v/v) glutaraldehyde was added and incubated at 4 C for 12 h to produce aldehyde groups on the enzymatic surfaces. The mixing solution was centrifuged at 9000 rpm for 20 min to remove unconjugated enzyme molecules and excess glutaraldehyde. The precipitate was resuspended in 10 mL of 10 mmol L-1 carbonate buffer (pH 8.6). Anti-AFP McAb (1 mg/mL, 5e30 mL) was added into the suspension solution under gentle stirring. The resultant solution was incubated for 4 h at 25 C. Then, 100 mL of 0.1 mol L-1 glycine was added, and the solution was incubated for another 1 h to seal the side residual
aldehydes. The probes were purified by centrifuging at 9000 rpm for 20 min to move unconjugated McAb and excess glycine. Following the removal of the supernatant, the red precipitate was washed once with PBS buffer by successive centrifugation and redispersion and then finally was suspended in 10 mL of PBS buffer (containing 1% BSA). The probes were then incubated at 4 C for 24 h to seal non-specific sites and increase the stability. The dispersity and diameters of the modified AuNPs were determined by a transmission electron microscopy (TEM, JEM-2100) and ultravioletvisible (UV-vis) spectroscopy (Cary 60). The hydrodynamic diameters of the modified AuNPs were analyzed by dynamic light scattering (DLS, LS-609). 2.5. Detection using ELISA The AuNP probe was synthesized using HRP and anti-AFP McAb (Fig. 1A). Compared with traditional McAbeHRP-based ELISA, the color density was amplified by the AuNP probe heavily loaded with HRP molecules (Fig. 1B). The assay was performed as follows: 100 mL of anti-AFP PcAb (10 mg mL-1) was added to microtiter plates. The plates were washed thrice with PBST after overnight incubation at 4 C. Then, 200 mL of 1% BSA was added to the plates, and the plates were incubated for another 1 h at 37 C. Another washing step was performed, and 50 mL of the sample solution and 50 mL of the AuNP probe were added to each well. The solutions were incubated at 37 C for 0.5 h. The plates were again washed to remove any unconjugated probe. Then, 100 mL of TMB solution was added, and the solutions were incubated for 5 min at room temperature. The colorimetric reaction was terminated by adding 50 mL of the stop solution. Absorbance was measured at 450 nm using a model 550 microplate reader (Bio-Rad). 3. Results and discussion 3.1. Characterization of AuNP probe In order to confirm that the probe was indeed successful synthesized, we test the particles during the synthesis process by TEM. The shape of bare AuNPs was spherical and monodispersed, and the average size was 17 nm in diameter (Fig. 2A). Fig. 2B shows HRP fixed to the AuNP surface, the protein halo around the AuNP indicated the successfully modification of AuNP. The TEM imagine of final probe was displayed in Fig. 2C, the probe was also in a colloid stability and monodispersion. Although the thick of protein halo was no obvious increased, this can be explained by considering that analysis by TEM the proteins chains are partially shrinkage during the sample drying process, and make the thin antibody membrane thinner. To further study the hydrodynamic diameters of bare AuNPs, HRP coated AuNPs and the final probes, DLS measurements were conducted (Fig. 2D). In the process of surface modification, the hydrodynamic diameters of particles was increased from 22 nm to 31 nm and then to 37 nm. The reasonable increase in the hydrodynamic diameters after the step-by-step modification process suggests that the proteins were effectively coupled with the particles. The particle size of the bare AuNPs detected by TEM (ca.17 nm) was smaller than that determined by DLS (22 nm) because of the size analysis by DLS is based on particle ensembles, and the AuNPs aggregate in the solution [20]. The DLS mode intensities yielded hydrodynamic diameters values that were systematically larger than those calculated by TEM in protein coated AuNPs. This phenomenon can be explained by considering that the proteins are partially extended under the conditions in DLS measurements. On the contrary, they are collapse in the dried state during TEM measurement [21,22]. Furthermore, both TEM and DLS results were in accord to show the AuNPs were successfully
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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Fig. 1. (A) Synthesis of gold nanoparticle (AuNP) probes and (B) principle of AuNP-based ELISA for highly sensitive detection of alpha fetoprotein (AFP).
Fig. 2. Transmission electron micrographs of bare AuNPs (A), HRP-AuNPs (B) and AuNP probes (C). (D) Size distribution of bare AuNPs (a), HRP-AuNPs (b), and McAb-HRP- AuNPs (c) were monitored by a DLS analyzer. (E) UV-vis spectra of bare AuNPs (a), HRP-AuNPs (b) and McAb-HRP-AuNPs (c).
modified by HRP and anti-AFP McAb through step-by-step modification process. Fig. 2E shows the UV-visible absorption spectra of bare AuNPs (a), HRP coated AuNPs (b) and the final probes(c). As shown, a strong absorption of bare AuNPs at 519 nm in aqueous solution. The absorption peak shifted from 519 nm to 522 nm and then to 524 nm with the layer-by-layer modification process. The result suggested that the protein was conjuncted to the particles and leads the increase in surface-coating thickness. The above observation confirmed that the antibody/HRP/AuNP complex was successful synthesis.
3.2. Optimal amount of anti-AFP McAb In the probe, HRP was employed as detector signal reagent and McAb was employed as detector antibody. The HRP was fixed to the AuNP surface in the probe via electrostatic and hydrophobic interactions [23]. The antibody was immobilized on the enzymatic surface by glutaraldehyde cross-linking. The amount of McAb is one of the most important components of the probe. Too little amount of antibodies results in false negative results, whereas excessive amounts may lead to false positives. Therefore, the amount of McAb
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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should be optimized. Fig. 3A shows that the absorbance value of the positive sample (2 mg/mL of AFP) increased with the volume of the antibody. However, the highest absorbance value was observed at 25 mL. Further increase in the volume of the antibody did not result in increased absorbance value. Therefore, the optimal amount of McAb was 25 mL which was used for further experiments.
3.3. Calibration curves To further demonstrate the capability of the synthesized probe on the ELISA, under optimal conditions, AFP standards with various concentrations were monitored in the 96-well microtiter plates by using the traditional ELISA protocol. For comparison, we also investigated the analytical properties of the conventional ELISA by directly using McAb-HRP as detection antibody. Optical signal was significantly enhanced using AuNP-based assays to detect AFP (Fig. 3A). This result was due to the higher amount of HRP carried on the AuNPs than that of McAb. Because the four-parameter logistic (4PL) model has been shown to be a useful and flexible tool in assaying the concentration of a single analyte for a variety ELISA formats [24,25], we employ the four-parameter logistic model to describe the sigmoidal relationship between absorbance value and log of AFP concentration. The 4PL model equation based on AuNP probe and McAb-HRP assays were y¼(2.88192e0.12718)/[1þ(x/
134.13132)1.39218]þ0.12718 with a coefficient correlation R2 ¼ 0.99587 and y¼(2.51563e0.11529)/[1þ(x/ 205.86554)2.09765]þ0.11529 with a coefficient correlation R2 ¼ 0.99162, respectively. The limit of detection (LOD) is determined by extrapolating the concentration at background level plus thrice the standard deviation (SD) of the background [26]. The horizontal dashed line marks the average blank response plus thrice the standard deviation. The intersection of horizontal dashed line and calibration curves indicates the LOD, and the LOD was determined as 2 ng/mL, which was more than 3 times lower that traditional McAb-HRP based ELISA (6 ng/mL). The low LOD was mainly ascribed to the higher amount of HRP carried on the AuNPs than on the McAb. 3.4. Cross-reactivity study To evaluate the selective of the dual-film modified probe, various proteins including alkaline phosphatase (ALP), osteopontin (OPN), ovalbumin (OVA) and BSA were test. AFP was used at concentration of 100 ng/mL while the solutions of other protein were prepared at concentration of 2 mg/mL with PBS and the blank was PBS. As shown in Fig. 4A, although the concentration of other tested proteins were higher than that of AFP, only AFP yielded significant signals, while signals from other proteins showed no significant difference from the blank control samples, thus demonstrating that AFP could be detected. AFP was diluted to 2 mg/mL with the four different sera (pig, horse, goat and bovine) to further evaluate the selectivity of the AuNP probe. As shown in Fig. 4B, significant signals were consistently observed. No statistically significant difference (F < F0.05) was found among the samples, thus indicating that the AuNP probe could be used to selectively detect AFP in the serum. Furthermore, we selected human sera that tested positive to AFP, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus to compare the different responses of the probe to different diseases. Only the AFP-positive serum yielded significant signals (Fig. 4C), and no cross-reactivity was observed with other sera that tested positive for the other viruses. This result indicates the effectiveness of the probe-based immunoassay to selectively detect AFP in human serum. 3.5. Detection of AFP in human serum To investigate whether this method was applicable to clinical diagnosis, we test the spiked normal human serum with three different concentrations of AFP: 20 ng/mL, 100 ng/mL, 200 ng/mL. The complex human serum with a mixture of proteins and other interfering substances didn't influence the AFP detection. As shown in Table 1, the average recovery of result was 97.95%, demonstrating that detection of AFP in human serum is feasible. Therefore, the proposed probe could be used for AFP detection in clinical diagnosis. 3.6. Stability of the probe The stability of the probe was evaluated using the procedure described in section 2.5, except that the AFP standard solution (2 mg/mL of AFP) was used instead of the sample solution. No obvious change in the absorbance value was observed in 21 days (Fig. 4D), thus indicating that the probe could be used for repeated measurements after three weeks of storage at 4 C.
Fig. 3. (A) Relationship between absorbance value and the volume of McAb. (B) Calibration curves for the absorbance value versus concentration of AFP. The horizontal dashed line marks the average blank response plus thrice the standard deviation (SD). Error bars, SD over three independent replicates.
4. Conclusions This study reported a signal-amplification strategy through dual-film modified AuNPs for the ultrasensitive detection of AFP in
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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Fig. 4. (A) Specificity of the probe. (B) Four different serum dilutions of AFP (2 mg/mL) using AuNP-based immunoassay. (C) Cross-reactivity studies against other diseases. (D) Stability of the dual-film-modified AuNP probe. No loss of activity was observed within 21 days of the probe preserved at 4 C.
Table 1 Recoveries of AFP from spiked human serum samples. Samplesa
Spiked concentration (ng/mL)
Detected concentrationb
Recovery
1 2 3
20 100 200
19.2 98.7 198.3
96 98.7 99.15
a Samples 1, 2, 3 were human serum samples spiked with three different concentrations of AFP: 20 ng/mL, 100 ng/mL, 200 ng/mL, respectively. b Each sample was analyzed three times, and the results are the average values.
the serum. HRP was fixed on the AuNP surface through a rapid and simple coating procedure. Glutaraldehyde cross-linking was performed to immobilize the McAb on the HRP. This novel strategy provides a simple and convenient process to synthesize McAbHRP-AuNP nanocomposites with colloidal stability and effective particle concentration. Comparison of the proposed probe and HRP-McAb-based immunoassay showed that the proposed strategy achieved an LOD of 2 ng/mL and a wide detection range with high specificity to AFP. The results suggest that dual-film modified AuNP-based immunoassay could be used for AFP detection in human serum. In addition, we believe this method may have a potential to detect other antigen by changing detector antibody. Acknowledgements This work is supported by National Natural Science Foundation of China (31270988, 31401577), China Postdoctoral Science Foundation (2014M562118) and Hunan Provincial Natural Science Foundation of China (2015JJ2133, 13JJ9004). References [1] Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistic. Ca A Cancer J Clin 2012;2015(65):87e108. [2] Tayob N, Lok AS, Do K-A, Feng Z. Improved detection of hepatocellular carcinoma by using a longitudinal alpha-fetoprotein screening algorithm. Clin
Gastroenterol Hepatol 2016;14:469e75. [3] Guo Z, Hao T, Duan J, Wang S, Wei D. Electrochemiluminescence immunosensor based on grapheneeCdS quantum dotseagarose composite for the ultrasensitive detection of alpha fetoprotein. Talanta 2012;89:27e32. [4] Kemp HA, Simpson J, Woodhead JS. Automated two-site immunoradiometric assay of human alpha-fetoprotein in maternal serum. Clin Chem 1981;27: 1388e91. [5] Huang C, Chen Y, Wang C, Zhu W, Ma H, Jin G. Detection of alpha-fetoprotein through biological signal amplification by biosensor based on imaging ellipsometry. Thin Solid Films 2011;519:2763e7. [6] Chang Y-F, Chen R-C, Lee Y-J, Chao S-C, Su L-C, Li Y-C, et al. Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum. Biosens Bioelectron 2009;24:1610e4. [7] Wu W, Li J, Pan D, Li J, Song S, Rong M, et al. Gold nanoparticle-based enzymelinked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium. Acs Appl Mater Interfaces 2014;6:16974e81. [8] Fenzl C, Hirsch T, Baeumner AJ. Nanomaterials as versatile tools for signal amplification in (bio) analytical applications. TrAC Trends Anal Chem 2016;79: 306e16. [9] Tang D, Cui Y, Chen G. Nanoparticle-based immunoassays in the biomedical field. Analyst 2013;138:981e90. [10] Edwards KA, Bolduc OR, Baeumner AJ. Miniaturized bioanalytical systems: enhanced performance through liposomes. Curr Opin Chem Biol 2012;16: 444e52. [11] Pei X, Zhang B, Tang J, Liu B, Lai W, Tang D. Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review. Anal Chim Acta 2013;758:1e18. [12] Quan K, Huang J, Yang X, Yang Y, Ying L, Wang H, et al. An enzyme-free and amplified colorimetric detection strategy: assembly of gold nanoparticles through target-catalytic circuits. Analyst 2015;140:1004e7. [13] Yockell-Lelievre H, Bukar N, McKeating K, Arnaud M, Cosin P, Guo Y, et al. Plasmonic sensors for the competitive detection of testosterone. Analyst 2015;140:5105e11. [14] Tan D, He Y, Xing X, Zhao Y, Tang H, Pang D. Aptamer functionalized gold nanoparticles based fluorescent probe for the detection of mercury (II) ion in aqueous solution. Talanta 2013;113:26e30. [15] Fournier-Wirth C, Coste J. Nanotechnologies for pathogen detection: future alternatives? Biologicals 2010;38:9e13. [16] Gao Z, Hou L, Xu M, Tang D. Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein. Sci Rep 2014;4:3966. [17] Li YS, Zhou Y, Meng XY, Zhang YY, Liu JQ, Zhang Y, et al. Enzymeeantibody dual labeled gold nanoparticles probe for ultrasensitive detection of k-casein in bovine milk samples. Biosens Bioelectron 2014;61:241e4. [18] Li YS, Meng XY, Zhou Y, Zhang YY, Meng XM, Yang L, et al. Magnetic bead and gold nanoparticle probes based immunoassay for b-casein detection in bovine milk samples. Biosens Bioelectron 2014;66:559e64.
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[19] Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature 1973;241:20e2. [20] Wei A, Mehtala JG, Patri AK. Challenges and opportunities in the advancement of nanomedicines. J Control Release 2012;164:236e46. [21] Lim Yb, Lee E, Lee M. Controlled bioactive nanostructures from self-assembly of peptide building blocks. Angew Chem Int Ed 2007;46:9011e4. [22] Walther A, Hoffmann M, Müller AH. Emulsion polymerization using Janus particles as stabilizers. Angew Chem 2008;120:723e6. [23] Cheung-Lau JC, Liu D, Pulsipher KW, Liu W, Dmochowski IJ. Engineering a well-ordered, functional protein-gold nanoparticle assembly. J Inorg Biochem
2014;130:59e68. [24] Jones G, Wortberg M, Kreissig SB, Bunch DS, Gee SJ, Hammock BD, et al. Extension of the four-parameter logistic model for ELISA to multianalyte analysis. J Immunol Methods 1995;177(1e2):1e7. [25] Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J physiol 1978;235(2):E97. [26] Rissin DM, Kan CW, Campbell TG, Howes SC, Fournier DR, Song L, et al. Singlemolecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010;28:595e9.
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein Qian Xiao, Yizhi Zheng, Jing Liu, Suiping Wang, Bo Feng* College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2016 Received in revised form 20 February 2017 Accepted 28 February 2017 Available online xxx
In this study, we designed a comprehensive strategy for the ultrasensitive detection of alpha fetoprotein (AFP) with high specificity using gold nanoparticle (AuNP)-based enzyme-linked immunosorbent assay (ELISA). A dual-film modified probe was synthesized by coating AuNPs with horseradish peroxidase (HRP) on its surface. Anti-AFP monoclonal antibody (McAb) was immobilized on the surface of the enzyme using glutaraldehyde cross-linking method. AuNPs, employed as support for the immobilization of HRP. HRP was used not only as the enzymatic-amplified tracer but also as a bridge for loading McAb. The limit of detection was 2 ng mL-1. The developed probes can provide an alternative approach with high sensitivity and a simple process similar to that of the traditional HRP-McAb based ELISA for the ultrasensitive detection of AFP in serum. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Gold nanoparticle Nanoprobe AFP ELISA
1. Introduction Serum alpha fetoprotein (AFP) has been extensively used as a biomarker for liver cancer, which is the second leading cause of cancer death in male worldwide [1]. Additionally, hepatocellular carcinoma (HCC) surveillance using ultrasound combined with AFP measurement is widely applied in practice. Strategies that enhance the sensitivity of existing surveillance tests can improve the feasibility of the early detection of HCC and may provide a more effective method for HCC surveillance [2]. Therefore, a sensitive detection and an accurate analysis of AFP concentrations are important. Various analytical techniques, such as electrochemiluminescence immunoassay [3], radioimmunoassay [4], imaging ellipsometry [5], and localized surface plasmon-coupled fluorescence [6] have been developed to determine AFP in human serum. These techniques are sensitive and highly specific for AFP detection. However, these methods are unsuitable for a low-cost, convenient, and rapid determination of AFP because they require skilled analysts and delicate instruments. ELISA, which is based on antibodyeantigen immunoreactions, is a powerful method in biological studies because of its convenient
* Corresponding author. Biological Engineering Department, College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China. E-mail address: [email protected] (B. Feng).
operation, capability of simultaneous measurement of large sample size, and low cost [7]. This approach is also the main method for measuring AFP. However, higher sensitivity is required for a precise diagnosis in clinical applications. Colloidal gold nanoparticles (AuNPs) have earned increasing attention in recent years in bioanalytical application including clinical diagnostics, point-of-care testing and therapeutic research as well as food safety and environmental monitoring [8]. One major merit of using AuNPs is that one can control and tailor their properties in a very predictable manner to meet the needs of specific application [9,10]. Therefore, AuNPs are extensively used for signal amplification in bioanalytical applications [11]. There are two main trends were well-established, one is utilizing the intrinsic properties of AuNPs such as color [12], localized surface plasmons [13], fluorescence resonance energy transfer [14], and many others. Another is carrying numerous biomolecules (e.g. antibody and enzyme molecule) and therefore generated a significant increase in signal [15]. Gao and co-workers designed a new ELISA strategy for highly sensitive colorimetric detection of prostate-specific antigen (PSA) by using AuNPs as the carriers of palladium nanostructures [16]. Li et al. synthesized a dual labeled probe by coating AuNPs with McAb and HRP on their surface. Based on the probe, an immunoassay was ultrasensitive detection of k-casein in bovine milk samples [17]. The same amplification technique also has been demonstrated in immunomagnetic beads separation [18]. In this study, AuNPs were employed to synthesize an ultrasensitive probe by dual-film assembly with HRP and anti-AFP
http://dx.doi.org/10.1016/j.biologicals.2017.02.008 1045-1056/© 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
2
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monoclonal antibody (McAb) on the AuNP surface. The introduction of dual-film modified AuNPs into ELISA resulted in an ultrasensitive signal amplification to quantitatively analyze AFP. 2. Materials and methods 2.1. Chemicals and reagents Chloroauric acid (HAuCl4) was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Polystyrene 96-well microtiter plates were obtained from Jet Bio-Filtration Products, Co., Ltd. (China). Mouse anti-AFP McAb was procured from Aoke Biotech Co., Ltd. (China). Rabbit anti-AFP polyclonal antibody (PcAb) was procured from Wuhan Huamei Biotech Co., Ltd. (China). HRPconjugated mouse anti-AFP antibody was obtained from SigmaAldrich (USA). AFP from human fetal cord serum and HRP were purchased from Sino Biological Inc. (China). 3,30 ,5,50 -tetramethylbenzidine (TMB), H2O2, and Tween-20 were supplied by Shanghai Macklin Biochemical Co., Ltd. (China). Alkaline phosphatase (ALP), osteopontin (OPN) and ovalbumin (OVA) were procured from Yeasen Biological Technology Co., Ltd. (China). The various sera were obtained from CellMax Co., Ltd. (China). All other chemicals were of analytical grade and obtained from Aladdin Co., Ltd. (China). 2.2. Solutions and buffers Phosphate-buffered saline (PBS, pH 7.4) containing 137 mmol LNaCl, 2.7 mmol L-1 KCl, 10 mmol L-1 Na2HPO4, and 2 mmol L1 KH2PO4. PBS solution with 0.05% Tween-20 (v/v) (PBST) was used as washing buffer. Coating buffer was 50 mmol L-1 carbonate buffer (pH 9.5). TMB solution consisted of 50 mmol L-1 sodium citrate buffer (pH 5.0) containing 0.01% (w/v) TMB and 0.005% (v/v) H2O2. The stop solution was 2 mol L-1 H2SO4. Deionized water (18 MU cm at 25 C) was used throughout the experiments. 1
2.3. Preparations of AuNPs AuNPs were prepared according to the chemical reduction method by Frens [19] with slight modification. All glassware was thoroughly cleaned in aqua regia [3v HCl/1v HNO3], rinsed in deionized water, and oven-dried prior to use. Then, 2 mL of 1% HAuCl4 solution in 200 mL of deionized water was boiled thoroughly with continuous stirring, and 4 mL of 1% trisodium citrate solution was quickly added under vigorous stirring. The solution color changed from gray to purple to wine-red. The solution was boiled for another 10 min. The heating source was removed, and the solution was continuously stirred for another 15 min. The solution was cooled to room temperature and stored in a dark bottle at 4 C. 2.4. Dual-film modified AuNPs The pH of AuNP solution was adjusted to 8.6 with 0.1 M Na2CO3. Then, 200 mL of 1 mg/mL HRP solution was added into 10 mL of the AuNP solution under agitation for 20 min. The solution was allowed to stand at 25 C without mixing for 2 h. Then, 100 mL of 1% (v/v) glutaraldehyde was added and incubated at 4 C for 12 h to produce aldehyde groups on the enzymatic surfaces. The mixing solution was centrifuged at 9000 rpm for 20 min to remove unconjugated enzyme molecules and excess glutaraldehyde. The precipitate was resuspended in 10 mL of 10 mmol L-1 carbonate buffer (pH 8.6). Anti-AFP McAb (1 mg/mL, 5e30 mL) was added into the suspension solution under gentle stirring. The resultant solution was incubated for 4 h at 25 C. Then, 100 mL of 0.1 mol L-1 glycine was added, and the solution was incubated for another 1 h to seal the side residual
aldehydes. The probes were purified by centrifuging at 9000 rpm for 20 min to move unconjugated McAb and excess glycine. Following the removal of the supernatant, the red precipitate was washed once with PBS buffer by successive centrifugation and redispersion and then finally was suspended in 10 mL of PBS buffer (containing 1% BSA). The probes were then incubated at 4 C for 24 h to seal non-specific sites and increase the stability. The dispersity and diameters of the modified AuNPs were determined by a transmission electron microscopy (TEM, JEM-2100) and ultravioletvisible (UV-vis) spectroscopy (Cary 60). The hydrodynamic diameters of the modified AuNPs were analyzed by dynamic light scattering (DLS, LS-609). 2.5. Detection using ELISA The AuNP probe was synthesized using HRP and anti-AFP McAb (Fig. 1A). Compared with traditional McAbeHRP-based ELISA, the color density was amplified by the AuNP probe heavily loaded with HRP molecules (Fig. 1B). The assay was performed as follows: 100 mL of anti-AFP PcAb (10 mg mL-1) was added to microtiter plates. The plates were washed thrice with PBST after overnight incubation at 4 C. Then, 200 mL of 1% BSA was added to the plates, and the plates were incubated for another 1 h at 37 C. Another washing step was performed, and 50 mL of the sample solution and 50 mL of the AuNP probe were added to each well. The solutions were incubated at 37 C for 0.5 h. The plates were again washed to remove any unconjugated probe. Then, 100 mL of TMB solution was added, and the solutions were incubated for 5 min at room temperature. The colorimetric reaction was terminated by adding 50 mL of the stop solution. Absorbance was measured at 450 nm using a model 550 microplate reader (Bio-Rad). 3. Results and discussion 3.1. Characterization of AuNP probe In order to confirm that the probe was indeed successful synthesized, we test the particles during the synthesis process by TEM. The shape of bare AuNPs was spherical and monodispersed, and the average size was 17 nm in diameter (Fig. 2A). Fig. 2B shows HRP fixed to the AuNP surface, the protein halo around the AuNP indicated the successfully modification of AuNP. The TEM imagine of final probe was displayed in Fig. 2C, the probe was also in a colloid stability and monodispersion. Although the thick of protein halo was no obvious increased, this can be explained by considering that analysis by TEM the proteins chains are partially shrinkage during the sample drying process, and make the thin antibody membrane thinner. To further study the hydrodynamic diameters of bare AuNPs, HRP coated AuNPs and the final probes, DLS measurements were conducted (Fig. 2D). In the process of surface modification, the hydrodynamic diameters of particles was increased from 22 nm to 31 nm and then to 37 nm. The reasonable increase in the hydrodynamic diameters after the step-by-step modification process suggests that the proteins were effectively coupled with the particles. The particle size of the bare AuNPs detected by TEM (ca.17 nm) was smaller than that determined by DLS (22 nm) because of the size analysis by DLS is based on particle ensembles, and the AuNPs aggregate in the solution [20]. The DLS mode intensities yielded hydrodynamic diameters values that were systematically larger than those calculated by TEM in protein coated AuNPs. This phenomenon can be explained by considering that the proteins are partially extended under the conditions in DLS measurements. On the contrary, they are collapse in the dried state during TEM measurement [21,22]. Furthermore, both TEM and DLS results were in accord to show the AuNPs were successfully
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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Fig. 1. (A) Synthesis of gold nanoparticle (AuNP) probes and (B) principle of AuNP-based ELISA for highly sensitive detection of alpha fetoprotein (AFP).
Fig. 2. Transmission electron micrographs of bare AuNPs (A), HRP-AuNPs (B) and AuNP probes (C). (D) Size distribution of bare AuNPs (a), HRP-AuNPs (b), and McAb-HRP- AuNPs (c) were monitored by a DLS analyzer. (E) UV-vis spectra of bare AuNPs (a), HRP-AuNPs (b) and McAb-HRP-AuNPs (c).
modified by HRP and anti-AFP McAb through step-by-step modification process. Fig. 2E shows the UV-visible absorption spectra of bare AuNPs (a), HRP coated AuNPs (b) and the final probes(c). As shown, a strong absorption of bare AuNPs at 519 nm in aqueous solution. The absorption peak shifted from 519 nm to 522 nm and then to 524 nm with the layer-by-layer modification process. The result suggested that the protein was conjuncted to the particles and leads the increase in surface-coating thickness. The above observation confirmed that the antibody/HRP/AuNP complex was successful synthesis.
3.2. Optimal amount of anti-AFP McAb In the probe, HRP was employed as detector signal reagent and McAb was employed as detector antibody. The HRP was fixed to the AuNP surface in the probe via electrostatic and hydrophobic interactions [23]. The antibody was immobilized on the enzymatic surface by glutaraldehyde cross-linking. The amount of McAb is one of the most important components of the probe. Too little amount of antibodies results in false negative results, whereas excessive amounts may lead to false positives. Therefore, the amount of McAb
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should be optimized. Fig. 3A shows that the absorbance value of the positive sample (2 mg/mL of AFP) increased with the volume of the antibody. However, the highest absorbance value was observed at 25 mL. Further increase in the volume of the antibody did not result in increased absorbance value. Therefore, the optimal amount of McAb was 25 mL which was used for further experiments.
3.3. Calibration curves To further demonstrate the capability of the synthesized probe on the ELISA, under optimal conditions, AFP standards with various concentrations were monitored in the 96-well microtiter plates by using the traditional ELISA protocol. For comparison, we also investigated the analytical properties of the conventional ELISA by directly using McAb-HRP as detection antibody. Optical signal was significantly enhanced using AuNP-based assays to detect AFP (Fig. 3A). This result was due to the higher amount of HRP carried on the AuNPs than that of McAb. Because the four-parameter logistic (4PL) model has been shown to be a useful and flexible tool in assaying the concentration of a single analyte for a variety ELISA formats [24,25], we employ the four-parameter logistic model to describe the sigmoidal relationship between absorbance value and log of AFP concentration. The 4PL model equation based on AuNP probe and McAb-HRP assays were y¼(2.88192e0.12718)/[1þ(x/
134.13132)1.39218]þ0.12718 with a coefficient correlation R2 ¼ 0.99587 and y¼(2.51563e0.11529)/[1þ(x/ 205.86554)2.09765]þ0.11529 with a coefficient correlation R2 ¼ 0.99162, respectively. The limit of detection (LOD) is determined by extrapolating the concentration at background level plus thrice the standard deviation (SD) of the background [26]. The horizontal dashed line marks the average blank response plus thrice the standard deviation. The intersection of horizontal dashed line and calibration curves indicates the LOD, and the LOD was determined as 2 ng/mL, which was more than 3 times lower that traditional McAb-HRP based ELISA (6 ng/mL). The low LOD was mainly ascribed to the higher amount of HRP carried on the AuNPs than on the McAb. 3.4. Cross-reactivity study To evaluate the selective of the dual-film modified probe, various proteins including alkaline phosphatase (ALP), osteopontin (OPN), ovalbumin (OVA) and BSA were test. AFP was used at concentration of 100 ng/mL while the solutions of other protein were prepared at concentration of 2 mg/mL with PBS and the blank was PBS. As shown in Fig. 4A, although the concentration of other tested proteins were higher than that of AFP, only AFP yielded significant signals, while signals from other proteins showed no significant difference from the blank control samples, thus demonstrating that AFP could be detected. AFP was diluted to 2 mg/mL with the four different sera (pig, horse, goat and bovine) to further evaluate the selectivity of the AuNP probe. As shown in Fig. 4B, significant signals were consistently observed. No statistically significant difference (F < F0.05) was found among the samples, thus indicating that the AuNP probe could be used to selectively detect AFP in the serum. Furthermore, we selected human sera that tested positive to AFP, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus to compare the different responses of the probe to different diseases. Only the AFP-positive serum yielded significant signals (Fig. 4C), and no cross-reactivity was observed with other sera that tested positive for the other viruses. This result indicates the effectiveness of the probe-based immunoassay to selectively detect AFP in human serum. 3.5. Detection of AFP in human serum To investigate whether this method was applicable to clinical diagnosis, we test the spiked normal human serum with three different concentrations of AFP: 20 ng/mL, 100 ng/mL, 200 ng/mL. The complex human serum with a mixture of proteins and other interfering substances didn't influence the AFP detection. As shown in Table 1, the average recovery of result was 97.95%, demonstrating that detection of AFP in human serum is feasible. Therefore, the proposed probe could be used for AFP detection in clinical diagnosis. 3.6. Stability of the probe The stability of the probe was evaluated using the procedure described in section 2.5, except that the AFP standard solution (2 mg/mL of AFP) was used instead of the sample solution. No obvious change in the absorbance value was observed in 21 days (Fig. 4D), thus indicating that the probe could be used for repeated measurements after three weeks of storage at 4 C.
Fig. 3. (A) Relationship between absorbance value and the volume of McAb. (B) Calibration curves for the absorbance value versus concentration of AFP. The horizontal dashed line marks the average blank response plus thrice the standard deviation (SD). Error bars, SD over three independent replicates.
4. Conclusions This study reported a signal-amplification strategy through dual-film modified AuNPs for the ultrasensitive detection of AFP in
Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008
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Fig. 4. (A) Specificity of the probe. (B) Four different serum dilutions of AFP (2 mg/mL) using AuNP-based immunoassay. (C) Cross-reactivity studies against other diseases. (D) Stability of the dual-film-modified AuNP probe. No loss of activity was observed within 21 days of the probe preserved at 4 C.
Table 1 Recoveries of AFP from spiked human serum samples. Samplesa
Spiked concentration (ng/mL)
Detected concentrationb
Recovery
1 2 3
20 100 200
19.2 98.7 198.3
96 98.7 99.15
a Samples 1, 2, 3 were human serum samples spiked with three different concentrations of AFP: 20 ng/mL, 100 ng/mL, 200 ng/mL, respectively. b Each sample was analyzed three times, and the results are the average values.
the serum. HRP was fixed on the AuNP surface through a rapid and simple coating procedure. Glutaraldehyde cross-linking was performed to immobilize the McAb on the HRP. This novel strategy provides a simple and convenient process to synthesize McAbHRP-AuNP nanocomposites with colloidal stability and effective particle concentration. Comparison of the proposed probe and HRP-McAb-based immunoassay showed that the proposed strategy achieved an LOD of 2 ng/mL and a wide detection range with high specificity to AFP. The results suggest that dual-film modified AuNP-based immunoassay could be used for AFP detection in human serum. In addition, we believe this method may have a potential to detect other antigen by changing detector antibody. Acknowledgements This work is supported by National Natural Science Foundation of China (31270988, 31401577), China Postdoctoral Science Foundation (2014M562118) and Hunan Provincial Natural Science Foundation of China (2015JJ2133, 13JJ9004). References [1] Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistic. Ca A Cancer J Clin 2012;2015(65):87e108. [2] Tayob N, Lok AS, Do K-A, Feng Z. Improved detection of hepatocellular carcinoma by using a longitudinal alpha-fetoprotein screening algorithm. Clin
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Please cite this article in press as: Xiao Q, et al., Enzymeeantibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.02.008