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A Modified ELISA Strategy Using Graphene Oxide Sheets and Gold Nanoparticles Functionalised with Different Antibody Types Hongjun Lin, Yingfu Liu, Jingrui Huo, Aihong Zhang, Yiting Pan, Haihong Bai, Jiao Zhang, Tian Fang, Xin Wang, Yun Cai, Qingming Wang, Yangjun Zhang, and Xiaohong Qian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401075u • Publication Date (Web): 28 May 2013 Downloaded from http://pubs.acs.org on May 30, 2013
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A Modified ELISA Strategy Using Graphene Oxide Sheets and Gold Nanoparticles Functionalised with Different Antibody Types† Hongjun Lin,#,†,‡ Yingfu Liu,#,†,‡ Jingrui Huo,†,‡ Aihong Zhang,§ Yiting Pan,†,‡ Haihong Bai,†,‡ Zhang Jiao,†,‡ Tian Fang,†,‡ Xin Wang,§Yun Cai,†,‡ Qingming Wang,†,‡ Yangjun Zhang*,†,‡ and Xiaohong Qian*,†,‡ 5
†
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing 102206, P. R. China Beijing Institute of Radiation Medicine, Beijing 100850, P. R. China § Institute of Chemical Defense, Beijing 102205, P. R. China ‡
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Gold nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent nano carriers in many analytical methods. In this study, a modified enzyme-linked immunosorbent assay (ELISA) strategy was developed using antibody-functionalised GO sheets and GNPs. This modification significantly reduced the limit of detection (LOD) and cost greatly of this assay. The applicability of the method was demonstrated by detecting HSP70 in a human
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serum sample. This result suggests that the 3G-ELISA method is feasible to detect an antigen in a complex mixture, and the LOD is up to 64-fold and the cost is as low as onetenth of the conventional ELISA method. c
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ELISA 1 is an effective and powerful method for protein detection and is the most commonly used analytical strategy for detecting and measuring trace biomarkers or other proteins. This method uses a horseradish peroxidase (HRP)-labelled immuno reagent to generate signal due to its low LOD,1 and standard operation procedures2,3 for this method have been available since its introduction in 1971 by Engvall and Perlmann4 and Van Weerman and Schuurs.5 Despite its popularity, ELISA still has some shortcomings, such as the need for expensive and large amounts of antibodies, a poor LOD, the requirement for multiple incubations for diffusion-limited reactions and the need for many washing steps followed by spectrophotometric detection using a chromogenic substrate. In addition, ELISA requires the use of expensive instruments and is labourious.6,7 To address these problems, some modifications have been made to ELISA, such as the introduction of GNPs and GO sheets.8-19 However, little progress has been reported towards amplifying the signal and reducing the cost of ELISA in any of the modified procedures that introduce GO and GNPs simultaneously. As excellent biological carriers, GNPs have many advantages, such as a high surface-to-volume ratio.20 In addition, GNPs are available in a wide range of sizes (1 to 200 nm)21 and can be easily conjugated to a variety of different proteins without affecting their biological activities.22 In this study, GNPs were prepared according to the classical method with slight modifications,23 and a GST-primary antibody-gold nanoparticlesassistant primary antibody (GST-Ab1-GNPs-GST-aAb1) was
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These authors contributed equally to this paper. * To whom correspondence should be addressed. E-mail:
[email protected],
[email protected] prepared by conjugating GNPs with Ab1 and aAb1 according to the previous studies.24,25 Graphene is a novel, one-atom thick, two-dimensional graphitic form of carbon that has drawn intense attention in analytical research because of its unique structure and easy conjugation to proteins without degrading their biological activity.26 In this research report, GO and horseradish peroxidaseimmunoglobulin G-graphene oxide (HRP-IgG-GO) were prepared using a modified Hummers method.27 On the basis of this work, we developed a novel ELISA strategy called 3G-ELISA by introducing solid complex GNPsGO-GNPs.
EXPERIMENTAL SECTION Materials and Instruments. Bovine serum albumin (BSA), 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 3, 30, 5, 50-tetramethyl-benzidine (TMB), 2-(Nmorpholino) ethanesulfonic acid (MES), Nhydroxysuccinimide (NHS), GST, GST-Ab1, cellular myelocytomatosis oncogene primary antibody (C-MYC-Ab1), HRP, IgG, Hsp70, Hsp70-Ab1, and Hsp70-aAb1were purchased from Sigma-Aldrich Chemical (Sigma-Aldrich, USA). Natural graphite powder (40 µm in size) was purchased from Qingdao Henglide Graphite Co. Ltd (Beijing, China). Chloroauric acid (HAuCl4·4H2O) and trisodium citrate were obtained from Shanghai Reagent Company (Shanghai, China). Deionized water (R > 18 MΩ) used for all experiments was purified by Millipore purification system (Shanghai, China). HSP70 was diluted in PBS (0.05 M, pH 7.0, by mixing the stock solutions of KH2PO4, Na2HPO4 and 0.1 M KCl), and PBST was PBS containing 0.05% (w/v) Tween 20. Blocking buffer solution is consisted of a PBS solution with added 2% (w/v) BSA (pH 7.4).
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Transmission electron microscopy (TEM) image was taken with a H-9000NAR instrument (HITACHI, Japan). X-ray photoelectron spectroscopy (XPS) measurements were done on a PHI Quantera Scanning X-ray Microprobe (ULVAC-PHI , Japan), which uses a focused monochromatic aluminum KR Xray (1486.7 eV) source for excitation and a spherical section analyzer. The samples were centrifuged by Sorvall Legend Micro 17 centrifuge (Thermo Scientific, USA). The optical density (OD) was obtained using Model 550 Microplat Reader (BIORAD, USA). Preparation of GNPs and aAb1-GNPs-Ab1. GNPs in our research were prepared according to the classical method with slight modifications. Briefly, first, all glassware used in experiment was thoroughly washed with aqua regia (three parts HCl, one part HNO3), rinsed in doubly distilled water, and ovendried prior to use. Second, 1 mL of 1% (mass percentage) HAuCl4·4H2O in 100mL doubly distilled water was brought to a boil at continuous stirring. Then 2.5 mL of l% (mass percentage) sodium citrate solution was quickly added, stirred, and kept boiling for another 15 min. The solution color changed from gray to blue, then purple, and finally to wine red during this period. Boiling was sustained for 10 min, then the heating source was removed, and the suspension was stirred for another 15 min, and stored in dark bottles at 4℃ for use.28,29 The conjugate of antibody-gold nanoparticles was prepared by addition of 30 µg Ab1 and aAb1 (C-MYC-Ab1, mole ratio is 1:10) to 1 mL suspension of gold nanoparticles (pH 6.0) followed by incubation at room temperature (RT) under stirring gently for 2 h, during which the Ab1 and aAb1 were adsorbed onto the GNPs through a combination of ionic and hydrophobic interactions. After blocked by 20 µL of 1% BSA solution for 30 min at RT, aAb1-GNPs-Ab1 was centrifuged at 13, 300 rpm for 10 min. A clear and pink supernatant of unbound antibody and a dark red, loosely packed sediment of the antibody-labeled GNPs were obtained. The supernatant was discarded and the sediment was rinsed with PBS. After another centrifugation at 13, 300 rpm for 10 min, the conjugate was redispersed in PBS containing 1% BSA added to increase stability of aAb1-GNPs-Ab1 and minimize nonspecific adsorption during the procedures. Finally, the conjugate was stored at 4 ℃ for use.30-32 The TEM images of GNPs and aAb1-GNPs-Ab1 were displayed in Figure S1 in the Supporting Information. The dark spots in Figure S1b are GNPs, and the floccus covering the dark spots are Ab1 and aAb1. Preparation of Ab2-GNPs-HRPs bioconjugates. GNPs were coated with Ab2 (IgG) and HRP according to a classical method. Briefly, first, 1.5 µL of 5.0 mg/mL Ab2 and 3.0 µL of 5.0 mg/mL HRP were added in 1.0 mL of the GNPs solution containing 0.04% trisodium citrate, 0.26 mM potassium carbonate, and 0.02% sodium azide. Second, after gently vortexed for 2 h, the mixture was blocked with 100 µL of 1% BSA solution for 30 min at RT, and then centrifuged at 15,000 rpm for 20 min at 4 ℃. After centrifugation, the product was washed with washing buffer, and the process was repeated three times. Finally, Ab2GNPs-HRPs bioconjugates were resuspended in 100 µL of 1% BSA for assay or stored at 4 ℃ when analysized later. The TEM
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images of GNPs and Ab2-GNPs-HRPs were displayed in Figure S2 in the Supporting Information. As shown in Figure S2, the dark spots in Figure S2b are GNPs, and the floccus covering the dark spots are Ab2 and HRPs. Preparation of GO and GO-Ab2-GNPs-HRPs. GO in our research was prepared using a modified Hummers method13-16 and graphene oxide-Ab2-GNPs-HRPs (GO-Ab2-GNPs-HRPs) was synthesized according to the literature17-19. Briefly, 50 mg of NaOH and ClCH2COONa were added to 1 mL of 1 mg mL-1 GO suspension, followed by bath sonication for 1.5 h. The resulting dispersion was washed 3 times with deionized water to remove the excess reagent. And then the GO was dispersed into 1 mL of pH 6.0 MES buffer containing 400 mM EDC and 200 mM NHS to give a homogeneous black suspension by 30 min activation. Excess EDC and NHS were discarded by centrifugation at 13, 300 rpm for 5 min, and the procedure was repeated for 3 times. Subsequently, the functionalized GO was dispersed in 1.0 mL of pH 7.4 PBS and the mixture was stirred for 4 h at RT. After centrifugation and wash for 3 times, the GO-Ab2-GNPs-HRPs conjugate was redispersed in 1.0 mL of PBS containing 1% BSA and stored at 4℃ for use.20-23 Figure S3a is the typical TEM image of samples GO sheets (Figure S3a) and GO-Ab2-GNPs-HRPs (Figure S3b). Lots of dark spots can be seen on GO sheets after conjugating with Ab2GNPs-HRPs. To ensure the dark spots did not originate from nonspecific absorption, the XPS detection was conducted. It can be seen that GO-Ab2-GNPs-HRPs image exhibits a single sharp N 1s spectrum centers at 399.6 eV (Curve a in Figure S4) while GO does not (Curve b in Figure S4), and this result indicates that the dark spots did originate from Ab2-GNPs-HRPs. Procedure for the modified 3G-ELISA. For those initial experiments, we used GST protein as a model antigen. In detail, the 96-well ELISA plates were coated with GST protein diluted in 100 µL carbonate buffer with pH 9.6 and incubated at 37 °C for 2 h. After three washes with PBST, the plates were sealed with 1% BSA, and incubated at 37 °C for 2 h or 4 °C overnight. After three washes with PBST, aAb1-GNPs-Ab1 was added and the plates were incubated at RT for 1 h. After three washes with PBST, GO-Ab2-GNPs-HRPs was added and plates were incubated at 37 °C for 30 min. After three washes, color was developed using the chromogen/substrate mixture TMB/H2O2. After 5 min again, the reaction was stopped by the addition of 2.0 M H2SO4. The OD of each well was read out at 450 nm using a microplate reader. The standard procedure of 3G-ELISA comprises the following steps shown in Figure 1. 1) immobilisation of an antigen in wells, 2) blocking the wells to prevent non-specific adsorption, 3) adding aAb1 and Ab1 to modify the GNPs, 4) washing away the unbound antibody, 5) adding the second antibody (Ab2) modified with GNPs-GO-HRP, 6) washing again, 7) adding the enzyme substrate and 8) measuring the optical density (OD).
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in this new method. To our knowledge, this is the first reported of a modified ELISA method that triples the signal amplification and reduces the cost in two ways through the introduction of GNPs and GO as carriers.
Figure 2. The schematic of the tripled signal amplification and dual cost reduction through the use of the 3G-ELISA method. 35
3G-ELISA has the following advantages over conventional ELISA (Table 1): 1) the LOD is reduced greatly from 64 ng/mL in conventional method to 1 ng/mL in 3G-ELISA and 2) the cost is reduced to nearly one-tenth of conventional ELISA. Figure 1. Schematic diagram of 3G-ELISA standard procedure.
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Table 1 Comparison of 3G-ELISA and conventional ELISA conventional 3G-ELISA ELISA
RESULTS AND DISCUSSION 5
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To examine the performance of the 3G-ELISA method, the following experiment was conducted, and GST protein was used as a model (Figure S5 in the Supporting Information). The LOD is 64 times lower with 3G-ELISA compared with conventional ELISA for GST protein determination. The improvement of the LOD for the 3G-ELISA strategy is the consequence of introducing GO sheets and GNPs as carriers for three amplifications. The GNPs conjugate several equivalents of Ab1 and aAb1; therefore, the ratio of Ab1 and aAb1 to GST ((Ab1 and aAb1)/GST) increases, which is referred to as the first amplification (Figure 2A). Then, GO sheets conjugate more Ab2, resulting in an increase in the ratio of Ab2 to Ab1 and aAb1 (Ab2/(Ab1 and aAb1)), which is the second amplification (Figure 2B). Finally, one Ab2 can bind more HRPs because the GNPs increase the ratio of HRPs/Ab2, which is called the third amplification (Figure 2C). In addition, cost reduction is another significant improvement with 3G-ELISA. First, a portion of Ab1 was replaced by aAb1 because of the lower price of aAb1 compared with Ab1 (Figure 2A). Second, more HRP is attached to Ab2 via the GNPs carrier to increase the ratio of HRP to Ab2 (Figure 2C). Because of the lower price of HRP than that of Ab2, the cost is further reduced
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primary antibody
aAb1s-GNPs-Ab1
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secondary antibody
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LOD
1 ng/mL
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low (about one-tenth of conventional ELISA)
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After verification of the 3G-ELISA method using GST as a model, we demonstrated the application of 3G-ELISA in the detection of HSP70.33-40 The Hsp70s are a family consisting of highly conserved protein molecular chaperones existing in most living organisms. These proteins play important roles in cell survival and the elimination of harmful effects, including heat shock, viral infection, exposure to chemical agents, osmosis, oxidation, hypothermia and toxicity, by preventing protein denaturation and participating in cell repair and scavenging of damaged proteins.41 The experimental procedures for this study are nearly the same as for the 3G-ELISA described above (Figure 3).
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positive patients as well as control samples (serum from normal human) using 3G-ELISA. Results were obtained for dilutions of human serum up to 64-fold. This result suggests that it is feasible to detect an antigen in a complex mixture, such as human serum, using 3G-ELISA.
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In summary, we have successfully developed a 3G-ELISA method with tripled signal amplification and dual cost reduction by the introduction of GO sheets and GNPs. The combination of two different primary antibodies (Ab1 and aAb1) attached to GNPs, a secondary antibody attached to GO sheets and HRP/Ab2 attached to GNPs significantly reduced the LOD and greatly reduced the cost. We believe that this concept can be exploited to widen its applicability for the analysis of trace biomarkers in complex mixtures. This work was financially supported by the National Key Program for Basic Research of China (2012CB910603 and 2010CB912704), The National Key Program for Scientific Instrument and Equipment Development (2011YQ030139), The National Program for high Technology Research and Development (2012AA020200) and The National Natural Science Foundation of China (21275159,21235001).
Notes and references
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Figure 3. Schematic diagram of a 3G-ELISA using antibody modified with
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Figure 4. Detection of HSP70 in human serum using 3G-ELISA. A) Schematic diagram of the antibody modified with 3G. B) Results of the assays for serum from patients and the control. C) Graph of the mean intensity of the colour in the well for detection. The heights of the bars represent the average 10
of eight independent measurements (N=8). The error bars represent one standard deviation from the average.
As shown in Figure 4, we measured HSP70 in a dilution series of serum samples from advanced-hepatocellular-carcinoma-
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