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Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay
Biosensors and Bioelectronics 53 (2014) 65–70
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay Xinle Jia, Xia Chen, Jingman Han, Jie Ma, Zhanfang Ma n Department of Chemistry, Capital Normal University, Beijing 100048, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2013 Received in revised form 7 September 2013 Accepted 10 September 2013 Available online 25 September 2013
Here we demonstrated an ultrasensitive electrochemical immunoassay employing graphene, platinum nanoparticles (PtNPs), glucose oxidase (GOD) and horseradish peroxidase (HRP) as enhancers to simultaneously detect carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). This immunosensor is based on the observation that multiple-labeled antibodies (thionine-labeled anti-CEA and ferrocenelabeled anti-AFP) recognition event yielded a distinct voltammetric peak through “sandwich” immunoreaction, whose position and size reflected the identity and level of the corresponding antigen. Greatly enhanced sensitivity for cancer markers is based on a triple signal amplification strategy. Experimental results revealed that the immunoassay enabled simultaneous determination of CEA and AFP in a single run with wide working ranges of 0.01–100 ng mL 1. The detection limits reached 1.64 pg mL 1 for CEA and 1.33 pg mL 1 for AFP. No obvious cross-talk was observed during the experiment. In addition, through the analysis of clinical serum samples, the proposed method received a good correlation with ELISA as a reference. The signal amplification strategy could be easily modified and extended to detect other multiple targets. & 2013 Elsevier B.V. All rights reserved.
Keywords: Graphene Signal amplification Simultaneous multianalyte detection Electrochemical immunoassay Tumor markers
1. Introduction Cancer is one of the deadliest diseases for human beings. The determination of tumor markers is great important in the early diagnosis and treatment of cancers (Kulasingam and Diamandis, 2008; Ferrari, 2005). While in clinical application, as most cancers have more than one marker associated with their incidence, the determination of a single tumor marker often limits diagnostic value (Mujagić et al., 2004; Kingsmore, 2006; Freedland, 2011). Therefore, the development of simple, reliable, powerful monitoring strategies for simultaneous determination of multiple tumor markers is particularly important in clinical laboratories. Recently the immunoassay for simultaneous determination of two or more tumor makers has been attracted much attention among the community (Chen et al., 2013; Wu et al., 2008; Chen et al., 2012; Lin and Ju, 2005). Among various measurement techniques, electrochemical immunoassay could be selected as an ideal strategy because of its portability, low cost and high sensitivity (Chikkaveeraiah et al., 2012; Li et al., 2012a). To date, the methodology to realize simultaneous multianalytes determination in electrochemistry is mainly based on spatial resolution
n
Corresponding author. Tel.: þ 86 1068902491. E-mail address: [email protected] (Z. Ma).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.09.021
(Lai et al., 2011; Leng et al., 2010) and multiple labels (Tang et al., 2011; Feng et al., 2012). The former mode usually requires specialized multi-working-electrode (sputter-deposited or screenprinted electrodes) or complex cutting tools to divide chips (Indium Tin Oxides) for capturing antibodies or antigens. For example, Lin et al. reported a label-free immunosensor based on modified mesoporous silica for simultaneous determination of tumor makers (Lin et al., 2011). Kong et al. introduced a branched electrode platform for simultaneous tumor makers detection based on different redox substrates (Kong et al., 2013). However, these electrodes are throwaway materials which make it be high cost and the detection sensitivity of these arrays is limited due to the nonenzymatic detection. In the latter mode, multiple-labeled antibodies are selectively bound onto the electrode surface through “sandwich” immunoreaction and the recognition event thus yield a distinct voltammetric peak, whose position and size reflect the identity and concentration of the corresponding antigens. This mode could achieve simultaneous multianalyte determination in a single run with convenient operation. Therefore, a simultaneous immunoassay for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) based on multiple labels was carried out in this work. Another key issue for a successful immunosensor is the signal amplification and noise reduction. New techniques using nanomaterials such as graphene (Lin et al., 2012; Liu et al., 2013a),
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magnetic particles (Peng et al., 2011), nanometallic materials (Yang et al., 2006, 2011), polymer membrane (Liu and Ma, 2013b) and enzyme (Hwang and Kim, 2005; Giannetto et al., 2011; Shang et al., 2013) are being developed to increase the sensitivity for cancer markers detection. For example, gold nanoparticles coated carbon nanotubes were used carriers to immobilize redox probe labeled antibodies for simultaneous determination of three liver cancer makers (Li et al., 2012b); Horseradish peroxidase (HRP) functionalized platinum hollow nanospheres were employed as probes for detecting CEA and AFP (Song et al., 2010); quantum dots (QDs) coated silica nanoparticles were used as labels for simultaneous detection of dual proteins by measuring the metallic component of QDs (Qian et al., 2011). However, there are two major drawbacks for these signal amplification techniques: (1) a nitrogen atmosphere for deoxygenation is necessary for removing the interference of dissolved oxygen in electrochemical assay based on HRP labels, one of the most popular labels in ELISA, which limits its clinical application; (2) harsh detection conditions including nanocrystals dissolution, high potential accumulation, and deoxygenation were required for QDs-based detection, which is not convenient for practical application. Herein, we designed an ultrasensitive electrochemical immunosensor employing AuNPs, bienzyme and platinum nanoparticles (PtNPs) functionalized graphene as enhancers to simultaneously detect CEA and AFP. To prepare the probes, thionine (THI) labeled anti-CEA and ferrocene (Fc) labeled anti-AFP were initially conjugated on PtNPs functionalized graphene nanocomposites, respectively, and the synthesized nanocomposites were then used as carriers for HRP and GOD. With the sandwich immunoassay format, the electrochemical signals were simultaneously obtained because of the presence of different electron mediators. Because the carried GOD and HRP catalyzed the oxidation of glucose and hydrogen peroxide, the electrochemical responses were enhanced. The peak currents and positions reflected the concentration and type of the corresponding antigens. Such a system well combines the simplicity of a single electrode platform and the signal amplification without deoxygenation. This assay approach could be modified and extended to the detection of other multiple targets.
2. Experimental 2.1. Reagents and materials Mouse monoclonal anti-CEA and anti-AFP, CEA and AFP was purchased from Linc-Bio Company (Shanghai, China). Human immunoglobulin G (IgG) was purchased from Chengwen Biological Company (Beijing, China). Graphene oxide was obtained from JCNANO (Nanjing, China). Chloroplatinic acid (H2PtCl6 6H2O), thionine acetate (THI), sodium borohydride (NaBH4), Ferrocenecarboxylic acid (Fc), Glucose, ascorbic acid (AA), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Alfa Aesar. Urea acid (UA) and albumin from bovine serum (BSA) were obtained from Beijing Chemical Reagents Company (Beijing, China). poly(ethylene imine) (PEI) was from Sigma-Aldrich. Clinical serum samples were available from Capital Normal University School Hospital. All the reagents were analytical grade and used as received.
measured on a CHI1140 electrochemical workstation (Shanghai, China). A three-electrode electrochemical cell was composed of a modified glass carbon electrode (GCE, Ø ¼4 mm) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. 2.3. Preparation of reduction graphene oxide (rGO) To prepare rGO, 10 mL stable dispersion of graphene oxide (1 mg mL 1) was mixed with 1 mL PEI (3%) and heated under reflux at 135 1C for 3 h. The obtained black dispersion was washed three times and collected by centrifugation. The final product was re-dispersed in 10 mL ultrapure water for further use. 2.4. Preparation of PtNPs functionalized graphene nanocomposites (PGN) The PGN were synthesized using NaBH4 reduction method. Briefly, 1 mL H2PtCl6 (1%) solution was added into 5 mL rGO dispersion and vigorously stirred for 5 min to make the negatively charged PtCl62 ions adsorbed on the rCO surface. Then, 2.5 mL NaBH4 solution was dropped into the mixture with stirring for 30 min. After several centrifugations and washings with Tris–HCl (pH¼9.0), the PGN was obtained and re-dispersed into ultrapure water prior to use. 2.5. Preparation of redox probe labeled antibodies, GOD and HRP functionalized PGN nanocomposites (PGN-Ab1/2) Firstly, EDC and NHS were used as coupling agents to modify the antibodies with redox probe by the formation of an amide link between the carboxyl of Fc and the amino of anti-AEP or between the amino of THI and the carboxyl of anti-CEA. The obtained redox probe labeled antibodies were centrifuged at 4 1C and washed several times with PBS (pH 7.3). Secondly, the modified antibodies were added into the PGN colloid followed by incubation at 4 1C for overnight. After centrifugation, the obtained PGN-Ab1/2 nanocomposites were totally washed three times with PBS (pH 7.3) to remove the uncombined redox probes. Finally, 1 mg GOD and 1 mg HRP were dissolved into the PGNAb1/2 (1 mg mL 1) to block the unspecified sites and prevent nonspecific adsorption. After several centrifugations and washing, the synthesized PGN-Ab1/2 carrying GOD and HRP were dispersed in PBS and store at 4 1C. 2.6. Preparation of multiple electrochemical immunoassay The GCE (Ø ¼4 mm) was polished repeatedly using alumina powder and then thoroughly cleaned before use. After that, the electrode was immersed in 20 mL 1% HAuCl4 aqueous solution and a constant potential of 0.2 V was applied for electrochemical deposition to obtain AuNPs. Then the GCE/AuNPs was soaked into the mixed solution containing 200 ng mL 1 anti-CEA and anti-AFP for 12 h at 4 1C, and BSA (1%) was employed to block possible remaining active sites and avoid the non-specific adsorption. After every step, the modified electrode was thoroughly cleaned with PBS. The as-prepared GCE/AuNPs/anntibodies were stored at 4 1C prior to use.
2.2. Apparatus 2.7. Electrochemical detection of CEA and AFP Images of the nanomaterials were taken via a Hitachi (H7650, 80 kV) transmission electron microscope. X-ray photoelectron spectroscopy (XPS) analysis was carried on an Escalab 250 X-ray Photoelectron Spectroscope (Thermofisher, American) using an Al (mono) Kα radiation. All electrochemical experiments were
All electrochemical measurements were performed at room temperature in 0.1 M pH 6.5 PBS solutions. The square wave voltammetry (SWV) scan was taken from 0.6 V to 0.6 V with a frequency of 15 Hz and a pulse amplitude of 25 mV (vs SCE). When
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Scheme 1. (A) Preparation procedure of PGN-Ab1/2 probes: (a) modification with PEI to obtain active groups of amino, (b) reducing H2PtCl6 to form PtNPs, (c) labeling PGN with thionine-anti-CEA, ferrocene-anti-AFP, HRP and GOD; (B) Schematic illustration of modification procedure of electrodes: (a) immobilization of anti-CEA (200 ng mL 1) and anti-AFP (200 ng mL 1) on GCE modified by AuNPs, (b) blocking possible remaining active sites and avoiding the non-specific adsorption with BSA (1%), (c) reaction with CEA and AFP; (C) Schematic illustration of the electrochemical immunoassay protocol and the measurement principle of the sandwich immunoassay.
measuring the detection, the as-prepared electrodes were successively incubated in various concentrations of antigens and the signal probes for 45 min at 37 1C, followed by washing with PBS. Then electrodes were placed into an electrochemical detector cell containing 4 mM glucose solutions to record the electrochemical signals.
3. Results and discussion 3.1. Principles of the electrochemical measurement In this work, we designed a universal electrochemical signal amplification strategy for simultaneous determination of CEA and AFP, using two distinguishable signal probes. As shown in Scheme 1A, thionine (THI) labeled anti-CEA and ferrocene (Fc) labeled anti-AFP were initially conjugated on PGN, respectively, and the synthesized nanocomposites were then used as carriers for HRP and GOD. Greatly enhanced sensitivity for cancer markers was based on a triple signal amplification strategy: (1) AuNPs modified on glassy carbon electrode (GCE) allowed several binding events of antibodies on each AuNP, which also greatly increased the electron transfer. (2) a large number of redox mediators labeled antibodies was immobilized on the GCE with the sandwich-type immunoassay format (Scheme 1B) and the signal was then amplified owing to the superior conductivity, surface area and catalytic performance of PtNPs functionalized graphene. (3) the GOD and HRP catalyzed the oxidation of glucose and hydrogen peroxide with the aid of the two independently mediators (Scheme 1C) to further enhance the current responses. The electrochemical signals were simultaneously obtained at various peak potentials. The peak currents and positions reflected the identity and level of the corresponding antigens. 3.2. Characterization of PGN and PGN-Ab1/2 nanocomposites The morphology and size of the synthesized nanocomposites were characterized by TEM. As shown in Fig. 1A, the graphene
oxide dispersion in water showed a homogeneous configuration and good dispersion. After the graphene oxide was reduced by PEI at 135 1C, the synthesized rGO (Fig. 1B) were surface-chemically modified to obtain abundant amino for attraction of nanoparticles. The dense and homogeneous coverage of PtNPs on the rGO can be observed clearly in Fig. 1C. Moreover, the synthesized graphene composites can homogeneously dispersed in water and the scraggy nanostructures provided a large surface area for the immobilization of biomolecules. To further analyze the composition of the nanomaterials, XPS characterization was employed (see Fig. S1 in the Supplementary Information). The characteristic peaks for C1s, N1s, O1s and Pt4f core-level regions could be obviously observed at the synthesized PGN (Fig. S1A). According to the literature (Bai et al., 2012), the Pt4f and C1s, N1s, O1s core levels were derived from the PtNPs and rGO, respectively. Fig. S1B and Fig. S1C displays the Pt4f, C1s, N1s, O1s and Fe2p3 or S2p core-level regions of PGN-Ab1/2. The existence of the Fe2p3 indicated the presence of Fc while the S2p core level was mainly derived from the redox probe THI. The results indicated that the PGN-Ab1/2 had been synthesized successfully. 3.3. Characterization of the multiplexed immunoassay In this work, the GCE electrodes were modified by layer-bylayer self-assembly method to construct stable immunosensor interfaces. It is very necessary to monitor the electrochemical behavior of the modification procedure by cyclic voltammetry (CV) and demonstrate the interfacial properties of physicochemical process in different modification-layers by electrochemical impedance spectroscopy (EIS). As shown in Fig. 2A, CV experiments were used to monitor the modification procedure of electrodes in 5 mM K3[Fe(CN)6]/K4[Fe (CN)6] solution containing 0.1 M KCl. The probe K3[Fe(CN)6]/K4[Fe (CN)6] reveals a reversible CV at the bare GCE (curve a). After the GCE was deposited with AuNPs, the peak current increased greatly
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Fig. 1. TEM images of (A) graphene oxide, (B) rGO and (C) PGN.
(curve b) due to the enhancement of AuNPs for electron transfer. When antibodies (curve c), BSA (curve d) and antigens (50 ng mL 1 CEA and AFP, curve e) were adsorbed on the AuNPs surfaces, the peak current successively decreased owing to the inhibition effect of the biomolecules for electron transfer. Significantly, the signal amplification performance of the sensor was monitored (Fig. 2B). After the as-prepared immunosensor was simultaneously incubated with PGN-Ab1/2 in 0.1 M PBS solution (pH 6.5), two pairs of redox waves for THI (at 0.15 V) and Fc (at þ 0.35 V) appeared (curve a), which could be distinguished clearly. After the addition of 4 mM glucose into the PBS, an obvious catalytic process was observed with the increase of cathodic peak and the decrease of anodic peak for the two couples of redox waves through the sandwich immunoreaction (curve b). The results indicated that the signal was greatly amplified because of excellent catalytic performance to glucose and H2O2. The electrochemical measurement and the electrocatalytic principle can be summarized as follows:
other biological molecules. Under the optimal conditions, the immunosensors were separately exposed to 10 mg mL 1 human IgG, 1 mg mL 1 glucose, 30 μg mL 1 UA and 1 mg mL 1 AA instead of 1 ng mL 1 CEA and AFP. As shown in Fig. S3 (see Fig. S3 in the Supplementary information), the current response for each interferent was weak and negligible compared with pure CEA and AFP. Stability is a key factor in the practical application for a successful immunosensor. When the immunosensor was stored in PBS, at pH 6.5 and 4 1C for 30 days, it retained 82.7% of its initial response. The above results suggested that the immunosensor showed excellent selectivity and stability. To investigate the reproducibility of immunosensor, five independently control tests were carried out to assay the same concentration of both tumor markers, receiving a relative standard deviation (RSD) of 6.2% for 1 ng mL 1 CEA and 4.9% for 1 ng mL 1 AFP. The lower RSD indicated that the immunosensor could be used repeatedly.
Glucose þ O2 ⟹GOD gluconate þ H 2 O2
3.5. Evaluation of the cross-talk for multiple immunoassays
HRP=Pt
H2 O2 ⟹ 2H þ þ 2e 1 þ O2 Thionineox =Ferroceneox þ 2e
1
þ
þ 2H -Thioninered =Ferrocenered
EIS was employed to probe the interfacial properties of modified electrodes. It is well known that the semicircle diameter in EIS equals to the electron transfer resistance (Ret) and the linear part at lower frequencies represents the diffusion process. As shown in Fig. 2C, with AuNPs assembled on the electrode surface, the Ret decreased (curve b, 89 Ω) than a bare GCE (curve a, 523 Ω). After the electrode was modified with antibodies, an increase in Ret was observed (curve c, 765 Ω) and a further increase was noticed (curve d, 943 Ω) when the GCE/AuNPs/Antibodies was blocked with BSA. Finally, the resistance (curve e, 1373 Ω) increased consecutively when the electrode was incubated with CEA and AFP, which was attributed to the obtained antigens that retarded the electron transfer tunnel. 3.4. Evaluation of selectivity, stability, reproducibility for multiple immunoassays Prior to experiment, the assay parameters including the pH of PBS (pH 6.5), the incubation temperature (37 1C) and time (45 min) for antibody-antigen immunoreaction were optimized (see Fig. S2 in the Supplementary information). The selectivity for the immunosensor was investigated with the present method. Here, we selected some possible interferents such as human IgG, glucose, UA and AA instead of CEA and AFP for the control experiments to further illustrate the non-specific absorption of
To evaluate the cross talk between the different analytes, another two control tests were conducted: (1) single analyte (1.0 and 50 ng mL 1 of CEA and AFP were used as examples), was assayed using the PGN-Ab1 and PGN-Ab2 as signal probes; (2) CEA and AFP were simultaneously monitored using the PGN-Ab1 and PGN-Ab2 as signal probes. Table S1 (see Table S1 in the Supplementary information) showed the electrochemical responses and the current shifts of the two control tests. Importantly, current response for the corresponding target analyte was higher while extremely low for another one. From the result we can determine that the multiplexed detection exhibited low interference between the two analytes and the cross-talk could be negligible. 3.6. Comparison of the multiple immunoassay using different probes and evaluation the validation of the amplification method To verify the advantages of the synthesized signal probes on the signal amplification for the multiplexed immunoassay, a comparative study was performed using two other different signal probes, redox probe labeled antibodies (THI-anti-CEA and Fc-antiAFP) and PtNPs (5 nm in diameter as an example) labeled the redox-antibodies (Pt-thionine-anti-CEA and Pt-ferrocene-antiAFP). Meanwhile, another control test was conducted to compare the current response in PBS with and without HRP and GOD. As indicated from Fig. 3, the current responses using PGN-Ab1/2 were greatly higher than the others. The immunosensors using redoxantibody (curve c) and Pt-redox-antibody (curve b) as signal tags exhibited low and smooth current changes at the low levels.
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Fig. 2. (A) CV characterization of the modified procedure of electrodes in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 M KCl: (a) bare GCE, (b) GCE/AuNPs, (c) GCE/ AuNPs/Antibodies, (d) GCE/AuNPs/Antibodies/BSA, (e) GCE/AuNPs/Antibodies/BSA/Antigens. (B) CV measurements using PGN-Ab1/2 as signal probes in 0.1 M PBS (a) and in 0.1 M PBS containing 4 mM GOD and HRP (b). (C) EIS characterization of the modified procedure of electrodes: (a) bare GCE, (b) GCE/AuNPs, (c) GCE/AuNPs/Antibodies, (d) GCE/AuNPs/Antibodies/BSA, and (e) GCE/AuNPs/Antibodies/BSA/Antigens.
Fig. 3. (A) Schematic illustration of the electrochemical immunoassay using different immunosensing probes: (a) PGN-Ab1/2, (b) Pt-Ab1/2, (c) labeled antibodies in 0.1 M PBS containing 4 mM GOD and HRP; Comparison of calibration curves of the electrochemical immunoassays for (B) CEA and (C) AFP. [Note: d is calibration curves for CEA and AFP in PBS which do not contain GOD and HRP.]
In addition, the present immunosensor exhibited a superior biosensing performance in PBS containing GOD and HRP (curve a) than that containing nothing (curve d). The presence of PGN not only obviously increased the surface area to capture a large amount of modified antibodies, and enzymes, but also accelerated the electron transfer. The immobilized GOD and HRP showed high catalytic efficiency with respect to glucose and H2O2. The result indicated that the immunosensor using PGN-Ab1/2 as signal probes exhibited high sensitivity as well as favored to detect CEA and AFP at very low concentrations. 3.7. Analytical performance of the multiplexed immunoassay To assess the performance of the proposed immunoassay, SWV measurements were carried out under the optimal experimental conditions after incubation with CEA and AFP standards and excess signal probes. As shown in Fig. 4A, the SWV reduction currents for THI at 1.5 V and for Fc at þ 3.5 V increased with an increase in the concentrations of CEA and AFP. The peak current of THI and Fc were separate proportional to the CEA (Fig. 4B) and AFP (Fig. 4C) concentrations in the working linear ranges of 0.01– 100 ng mL 1. The correlation coefficients were 0.998 and 0.994, respectively. The detection limits reached 1.64 pg mL 1 for CEA and 1.33 pg mL 1 for AFP at a signal-to-noise ratio of 3. The comparison of the working ranges and detection limits between the proposed biosensor and some recently published studies was listed in Table S2 (see Table S2 in the Supplementary information). The result indicated that the proposed electrochemical immunoassay using the triple signal amplification enabled relatively wide linear ranges and low LODs.
3.8. Analysis of clinical serum samples To investigate the analytical reliability and application potential of the proposed immunosensing method, ten human serum samples from Hospital of Capital Normal University (Beijing, China) were directly used for assay without dilution using the present method. The results using an enzyme-linked immunosorbent assay (ELISA) as a reference method were shown in Table S3 (see Table S3 in the Supplementary information). The relative errors between the two methods were both from 10% to 10% for CEA and AFP, indicating good accuracy of the proposed method for sample assay.
4. Conclusion In this work, we have successfully developed a novel ultrasensitive multiplexed electrochemical immunoassay for simultaneous detection of CEA and AFP using biofunctionalized graphene nanocomposites as distinguishable signal probes. The immunosensor with high sensitivity and wide linear ranges was achieved because of the performance of AuNPs and excellent electrocatalysis activities of the biofunctionalized graphene nanocomposites. Compared with other assays, highlights of this work can be summarized as follows: (1) A triple signal amplification strategy was constructed for ultrasensitive electrochemistry detection of CEA and AFP. (2) The immunosensor received a good correlation with ELISA as a reference. The multiple immunoassays with high selectivity, good stability and reproducibility could be readily applied in clinical diagnosis. (3) Such an immunosensor well
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Fig. 4. SWV responses (A) and calibration curves for different concentrations of CEA (B) and AFP (C) in PBS, pH 6.5, containing 4 mM GOD and HRP.
combines the simplicity of a single working-electrode platform and the amplified signal response without deoxygenation for simultaneous multianalyte determination in a single run. It can be foreseen that the universal method demonstrated here could be modified and extended to the detection of other multiple targets. Acknowledgments This research was financed by grants from the National Natural Science Foundation of China (21273153) and Beijing Natural Science Foundation (2132008).
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Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay Xinle Jia, Xia Chen, Jingman Han, Jie Ma, Zhanfang Ma n Department of Chemistry, Capital Normal University, Beijing 100048, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2013 Received in revised form 7 September 2013 Accepted 10 September 2013 Available online 25 September 2013
Here we demonstrated an ultrasensitive electrochemical immunoassay employing graphene, platinum nanoparticles (PtNPs), glucose oxidase (GOD) and horseradish peroxidase (HRP) as enhancers to simultaneously detect carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). This immunosensor is based on the observation that multiple-labeled antibodies (thionine-labeled anti-CEA and ferrocenelabeled anti-AFP) recognition event yielded a distinct voltammetric peak through “sandwich” immunoreaction, whose position and size reflected the identity and level of the corresponding antigen. Greatly enhanced sensitivity for cancer markers is based on a triple signal amplification strategy. Experimental results revealed that the immunoassay enabled simultaneous determination of CEA and AFP in a single run with wide working ranges of 0.01–100 ng mL 1. The detection limits reached 1.64 pg mL 1 for CEA and 1.33 pg mL 1 for AFP. No obvious cross-talk was observed during the experiment. In addition, through the analysis of clinical serum samples, the proposed method received a good correlation with ELISA as a reference. The signal amplification strategy could be easily modified and extended to detect other multiple targets. & 2013 Elsevier B.V. All rights reserved.
Keywords: Graphene Signal amplification Simultaneous multianalyte detection Electrochemical immunoassay Tumor markers
1. Introduction Cancer is one of the deadliest diseases for human beings. The determination of tumor markers is great important in the early diagnosis and treatment of cancers (Kulasingam and Diamandis, 2008; Ferrari, 2005). While in clinical application, as most cancers have more than one marker associated with their incidence, the determination of a single tumor marker often limits diagnostic value (Mujagić et al., 2004; Kingsmore, 2006; Freedland, 2011). Therefore, the development of simple, reliable, powerful monitoring strategies for simultaneous determination of multiple tumor markers is particularly important in clinical laboratories. Recently the immunoassay for simultaneous determination of two or more tumor makers has been attracted much attention among the community (Chen et al., 2013; Wu et al., 2008; Chen et al., 2012; Lin and Ju, 2005). Among various measurement techniques, electrochemical immunoassay could be selected as an ideal strategy because of its portability, low cost and high sensitivity (Chikkaveeraiah et al., 2012; Li et al., 2012a). To date, the methodology to realize simultaneous multianalytes determination in electrochemistry is mainly based on spatial resolution
n
Corresponding author. Tel.: þ 86 1068902491. E-mail address: [email protected] (Z. Ma).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.09.021
(Lai et al., 2011; Leng et al., 2010) and multiple labels (Tang et al., 2011; Feng et al., 2012). The former mode usually requires specialized multi-working-electrode (sputter-deposited or screenprinted electrodes) or complex cutting tools to divide chips (Indium Tin Oxides) for capturing antibodies or antigens. For example, Lin et al. reported a label-free immunosensor based on modified mesoporous silica for simultaneous determination of tumor makers (Lin et al., 2011). Kong et al. introduced a branched electrode platform for simultaneous tumor makers detection based on different redox substrates (Kong et al., 2013). However, these electrodes are throwaway materials which make it be high cost and the detection sensitivity of these arrays is limited due to the nonenzymatic detection. In the latter mode, multiple-labeled antibodies are selectively bound onto the electrode surface through “sandwich” immunoreaction and the recognition event thus yield a distinct voltammetric peak, whose position and size reflect the identity and concentration of the corresponding antigens. This mode could achieve simultaneous multianalyte determination in a single run with convenient operation. Therefore, a simultaneous immunoassay for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) based on multiple labels was carried out in this work. Another key issue for a successful immunosensor is the signal amplification and noise reduction. New techniques using nanomaterials such as graphene (Lin et al., 2012; Liu et al., 2013a),
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magnetic particles (Peng et al., 2011), nanometallic materials (Yang et al., 2006, 2011), polymer membrane (Liu and Ma, 2013b) and enzyme (Hwang and Kim, 2005; Giannetto et al., 2011; Shang et al., 2013) are being developed to increase the sensitivity for cancer markers detection. For example, gold nanoparticles coated carbon nanotubes were used carriers to immobilize redox probe labeled antibodies for simultaneous determination of three liver cancer makers (Li et al., 2012b); Horseradish peroxidase (HRP) functionalized platinum hollow nanospheres were employed as probes for detecting CEA and AFP (Song et al., 2010); quantum dots (QDs) coated silica nanoparticles were used as labels for simultaneous detection of dual proteins by measuring the metallic component of QDs (Qian et al., 2011). However, there are two major drawbacks for these signal amplification techniques: (1) a nitrogen atmosphere for deoxygenation is necessary for removing the interference of dissolved oxygen in electrochemical assay based on HRP labels, one of the most popular labels in ELISA, which limits its clinical application; (2) harsh detection conditions including nanocrystals dissolution, high potential accumulation, and deoxygenation were required for QDs-based detection, which is not convenient for practical application. Herein, we designed an ultrasensitive electrochemical immunosensor employing AuNPs, bienzyme and platinum nanoparticles (PtNPs) functionalized graphene as enhancers to simultaneously detect CEA and AFP. To prepare the probes, thionine (THI) labeled anti-CEA and ferrocene (Fc) labeled anti-AFP were initially conjugated on PtNPs functionalized graphene nanocomposites, respectively, and the synthesized nanocomposites were then used as carriers for HRP and GOD. With the sandwich immunoassay format, the electrochemical signals were simultaneously obtained because of the presence of different electron mediators. Because the carried GOD and HRP catalyzed the oxidation of glucose and hydrogen peroxide, the electrochemical responses were enhanced. The peak currents and positions reflected the concentration and type of the corresponding antigens. Such a system well combines the simplicity of a single electrode platform and the signal amplification without deoxygenation. This assay approach could be modified and extended to the detection of other multiple targets.
2. Experimental 2.1. Reagents and materials Mouse monoclonal anti-CEA and anti-AFP, CEA and AFP was purchased from Linc-Bio Company (Shanghai, China). Human immunoglobulin G (IgG) was purchased from Chengwen Biological Company (Beijing, China). Graphene oxide was obtained from JCNANO (Nanjing, China). Chloroplatinic acid (H2PtCl6 6H2O), thionine acetate (THI), sodium borohydride (NaBH4), Ferrocenecarboxylic acid (Fc), Glucose, ascorbic acid (AA), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Alfa Aesar. Urea acid (UA) and albumin from bovine serum (BSA) were obtained from Beijing Chemical Reagents Company (Beijing, China). poly(ethylene imine) (PEI) was from Sigma-Aldrich. Clinical serum samples were available from Capital Normal University School Hospital. All the reagents were analytical grade and used as received.
measured on a CHI1140 electrochemical workstation (Shanghai, China). A three-electrode electrochemical cell was composed of a modified glass carbon electrode (GCE, Ø ¼4 mm) as the working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode (SCE) as the reference electrode. 2.3. Preparation of reduction graphene oxide (rGO) To prepare rGO, 10 mL stable dispersion of graphene oxide (1 mg mL 1) was mixed with 1 mL PEI (3%) and heated under reflux at 135 1C for 3 h. The obtained black dispersion was washed three times and collected by centrifugation. The final product was re-dispersed in 10 mL ultrapure water for further use. 2.4. Preparation of PtNPs functionalized graphene nanocomposites (PGN) The PGN were synthesized using NaBH4 reduction method. Briefly, 1 mL H2PtCl6 (1%) solution was added into 5 mL rGO dispersion and vigorously stirred for 5 min to make the negatively charged PtCl62 ions adsorbed on the rCO surface. Then, 2.5 mL NaBH4 solution was dropped into the mixture with stirring for 30 min. After several centrifugations and washings with Tris–HCl (pH¼9.0), the PGN was obtained and re-dispersed into ultrapure water prior to use. 2.5. Preparation of redox probe labeled antibodies, GOD and HRP functionalized PGN nanocomposites (PGN-Ab1/2) Firstly, EDC and NHS were used as coupling agents to modify the antibodies with redox probe by the formation of an amide link between the carboxyl of Fc and the amino of anti-AEP or between the amino of THI and the carboxyl of anti-CEA. The obtained redox probe labeled antibodies were centrifuged at 4 1C and washed several times with PBS (pH 7.3). Secondly, the modified antibodies were added into the PGN colloid followed by incubation at 4 1C for overnight. After centrifugation, the obtained PGN-Ab1/2 nanocomposites were totally washed three times with PBS (pH 7.3) to remove the uncombined redox probes. Finally, 1 mg GOD and 1 mg HRP were dissolved into the PGNAb1/2 (1 mg mL 1) to block the unspecified sites and prevent nonspecific adsorption. After several centrifugations and washing, the synthesized PGN-Ab1/2 carrying GOD and HRP were dispersed in PBS and store at 4 1C. 2.6. Preparation of multiple electrochemical immunoassay The GCE (Ø ¼4 mm) was polished repeatedly using alumina powder and then thoroughly cleaned before use. After that, the electrode was immersed in 20 mL 1% HAuCl4 aqueous solution and a constant potential of 0.2 V was applied for electrochemical deposition to obtain AuNPs. Then the GCE/AuNPs was soaked into the mixed solution containing 200 ng mL 1 anti-CEA and anti-AFP for 12 h at 4 1C, and BSA (1%) was employed to block possible remaining active sites and avoid the non-specific adsorption. After every step, the modified electrode was thoroughly cleaned with PBS. The as-prepared GCE/AuNPs/anntibodies were stored at 4 1C prior to use.
2.2. Apparatus 2.7. Electrochemical detection of CEA and AFP Images of the nanomaterials were taken via a Hitachi (H7650, 80 kV) transmission electron microscope. X-ray photoelectron spectroscopy (XPS) analysis was carried on an Escalab 250 X-ray Photoelectron Spectroscope (Thermofisher, American) using an Al (mono) Kα radiation. All electrochemical experiments were
All electrochemical measurements were performed at room temperature in 0.1 M pH 6.5 PBS solutions. The square wave voltammetry (SWV) scan was taken from 0.6 V to 0.6 V with a frequency of 15 Hz and a pulse amplitude of 25 mV (vs SCE). When
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Scheme 1. (A) Preparation procedure of PGN-Ab1/2 probes: (a) modification with PEI to obtain active groups of amino, (b) reducing H2PtCl6 to form PtNPs, (c) labeling PGN with thionine-anti-CEA, ferrocene-anti-AFP, HRP and GOD; (B) Schematic illustration of modification procedure of electrodes: (a) immobilization of anti-CEA (200 ng mL 1) and anti-AFP (200 ng mL 1) on GCE modified by AuNPs, (b) blocking possible remaining active sites and avoiding the non-specific adsorption with BSA (1%), (c) reaction with CEA and AFP; (C) Schematic illustration of the electrochemical immunoassay protocol and the measurement principle of the sandwich immunoassay.
measuring the detection, the as-prepared electrodes were successively incubated in various concentrations of antigens and the signal probes for 45 min at 37 1C, followed by washing with PBS. Then electrodes were placed into an electrochemical detector cell containing 4 mM glucose solutions to record the electrochemical signals.
3. Results and discussion 3.1. Principles of the electrochemical measurement In this work, we designed a universal electrochemical signal amplification strategy for simultaneous determination of CEA and AFP, using two distinguishable signal probes. As shown in Scheme 1A, thionine (THI) labeled anti-CEA and ferrocene (Fc) labeled anti-AFP were initially conjugated on PGN, respectively, and the synthesized nanocomposites were then used as carriers for HRP and GOD. Greatly enhanced sensitivity for cancer markers was based on a triple signal amplification strategy: (1) AuNPs modified on glassy carbon electrode (GCE) allowed several binding events of antibodies on each AuNP, which also greatly increased the electron transfer. (2) a large number of redox mediators labeled antibodies was immobilized on the GCE with the sandwich-type immunoassay format (Scheme 1B) and the signal was then amplified owing to the superior conductivity, surface area and catalytic performance of PtNPs functionalized graphene. (3) the GOD and HRP catalyzed the oxidation of glucose and hydrogen peroxide with the aid of the two independently mediators (Scheme 1C) to further enhance the current responses. The electrochemical signals were simultaneously obtained at various peak potentials. The peak currents and positions reflected the identity and level of the corresponding antigens. 3.2. Characterization of PGN and PGN-Ab1/2 nanocomposites The morphology and size of the synthesized nanocomposites were characterized by TEM. As shown in Fig. 1A, the graphene
oxide dispersion in water showed a homogeneous configuration and good dispersion. After the graphene oxide was reduced by PEI at 135 1C, the synthesized rGO (Fig. 1B) were surface-chemically modified to obtain abundant amino for attraction of nanoparticles. The dense and homogeneous coverage of PtNPs on the rGO can be observed clearly in Fig. 1C. Moreover, the synthesized graphene composites can homogeneously dispersed in water and the scraggy nanostructures provided a large surface area for the immobilization of biomolecules. To further analyze the composition of the nanomaterials, XPS characterization was employed (see Fig. S1 in the Supplementary Information). The characteristic peaks for C1s, N1s, O1s and Pt4f core-level regions could be obviously observed at the synthesized PGN (Fig. S1A). According to the literature (Bai et al., 2012), the Pt4f and C1s, N1s, O1s core levels were derived from the PtNPs and rGO, respectively. Fig. S1B and Fig. S1C displays the Pt4f, C1s, N1s, O1s and Fe2p3 or S2p core-level regions of PGN-Ab1/2. The existence of the Fe2p3 indicated the presence of Fc while the S2p core level was mainly derived from the redox probe THI. The results indicated that the PGN-Ab1/2 had been synthesized successfully. 3.3. Characterization of the multiplexed immunoassay In this work, the GCE electrodes were modified by layer-bylayer self-assembly method to construct stable immunosensor interfaces. It is very necessary to monitor the electrochemical behavior of the modification procedure by cyclic voltammetry (CV) and demonstrate the interfacial properties of physicochemical process in different modification-layers by electrochemical impedance spectroscopy (EIS). As shown in Fig. 2A, CV experiments were used to monitor the modification procedure of electrodes in 5 mM K3[Fe(CN)6]/K4[Fe (CN)6] solution containing 0.1 M KCl. The probe K3[Fe(CN)6]/K4[Fe (CN)6] reveals a reversible CV at the bare GCE (curve a). After the GCE was deposited with AuNPs, the peak current increased greatly
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Fig. 1. TEM images of (A) graphene oxide, (B) rGO and (C) PGN.
(curve b) due to the enhancement of AuNPs for electron transfer. When antibodies (curve c), BSA (curve d) and antigens (50 ng mL 1 CEA and AFP, curve e) were adsorbed on the AuNPs surfaces, the peak current successively decreased owing to the inhibition effect of the biomolecules for electron transfer. Significantly, the signal amplification performance of the sensor was monitored (Fig. 2B). After the as-prepared immunosensor was simultaneously incubated with PGN-Ab1/2 in 0.1 M PBS solution (pH 6.5), two pairs of redox waves for THI (at 0.15 V) and Fc (at þ 0.35 V) appeared (curve a), which could be distinguished clearly. After the addition of 4 mM glucose into the PBS, an obvious catalytic process was observed with the increase of cathodic peak and the decrease of anodic peak for the two couples of redox waves through the sandwich immunoreaction (curve b). The results indicated that the signal was greatly amplified because of excellent catalytic performance to glucose and H2O2. The electrochemical measurement and the electrocatalytic principle can be summarized as follows:
other biological molecules. Under the optimal conditions, the immunosensors were separately exposed to 10 mg mL 1 human IgG, 1 mg mL 1 glucose, 30 μg mL 1 UA and 1 mg mL 1 AA instead of 1 ng mL 1 CEA and AFP. As shown in Fig. S3 (see Fig. S3 in the Supplementary information), the current response for each interferent was weak and negligible compared with pure CEA and AFP. Stability is a key factor in the practical application for a successful immunosensor. When the immunosensor was stored in PBS, at pH 6.5 and 4 1C for 30 days, it retained 82.7% of its initial response. The above results suggested that the immunosensor showed excellent selectivity and stability. To investigate the reproducibility of immunosensor, five independently control tests were carried out to assay the same concentration of both tumor markers, receiving a relative standard deviation (RSD) of 6.2% for 1 ng mL 1 CEA and 4.9% for 1 ng mL 1 AFP. The lower RSD indicated that the immunosensor could be used repeatedly.
Glucose þ O2 ⟹GOD gluconate þ H 2 O2
3.5. Evaluation of the cross-talk for multiple immunoassays
HRP=Pt
H2 O2 ⟹ 2H þ þ 2e 1 þ O2 Thionineox =Ferroceneox þ 2e
1
þ
þ 2H -Thioninered =Ferrocenered
EIS was employed to probe the interfacial properties of modified electrodes. It is well known that the semicircle diameter in EIS equals to the electron transfer resistance (Ret) and the linear part at lower frequencies represents the diffusion process. As shown in Fig. 2C, with AuNPs assembled on the electrode surface, the Ret decreased (curve b, 89 Ω) than a bare GCE (curve a, 523 Ω). After the electrode was modified with antibodies, an increase in Ret was observed (curve c, 765 Ω) and a further increase was noticed (curve d, 943 Ω) when the GCE/AuNPs/Antibodies was blocked with BSA. Finally, the resistance (curve e, 1373 Ω) increased consecutively when the electrode was incubated with CEA and AFP, which was attributed to the obtained antigens that retarded the electron transfer tunnel. 3.4. Evaluation of selectivity, stability, reproducibility for multiple immunoassays Prior to experiment, the assay parameters including the pH of PBS (pH 6.5), the incubation temperature (37 1C) and time (45 min) for antibody-antigen immunoreaction were optimized (see Fig. S2 in the Supplementary information). The selectivity for the immunosensor was investigated with the present method. Here, we selected some possible interferents such as human IgG, glucose, UA and AA instead of CEA and AFP for the control experiments to further illustrate the non-specific absorption of
To evaluate the cross talk between the different analytes, another two control tests were conducted: (1) single analyte (1.0 and 50 ng mL 1 of CEA and AFP were used as examples), was assayed using the PGN-Ab1 and PGN-Ab2 as signal probes; (2) CEA and AFP were simultaneously monitored using the PGN-Ab1 and PGN-Ab2 as signal probes. Table S1 (see Table S1 in the Supplementary information) showed the electrochemical responses and the current shifts of the two control tests. Importantly, current response for the corresponding target analyte was higher while extremely low for another one. From the result we can determine that the multiplexed detection exhibited low interference between the two analytes and the cross-talk could be negligible. 3.6. Comparison of the multiple immunoassay using different probes and evaluation the validation of the amplification method To verify the advantages of the synthesized signal probes on the signal amplification for the multiplexed immunoassay, a comparative study was performed using two other different signal probes, redox probe labeled antibodies (THI-anti-CEA and Fc-antiAFP) and PtNPs (5 nm in diameter as an example) labeled the redox-antibodies (Pt-thionine-anti-CEA and Pt-ferrocene-antiAFP). Meanwhile, another control test was conducted to compare the current response in PBS with and without HRP and GOD. As indicated from Fig. 3, the current responses using PGN-Ab1/2 were greatly higher than the others. The immunosensors using redoxantibody (curve c) and Pt-redox-antibody (curve b) as signal tags exhibited low and smooth current changes at the low levels.
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Fig. 2. (A) CV characterization of the modified procedure of electrodes in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 M KCl: (a) bare GCE, (b) GCE/AuNPs, (c) GCE/ AuNPs/Antibodies, (d) GCE/AuNPs/Antibodies/BSA, (e) GCE/AuNPs/Antibodies/BSA/Antigens. (B) CV measurements using PGN-Ab1/2 as signal probes in 0.1 M PBS (a) and in 0.1 M PBS containing 4 mM GOD and HRP (b). (C) EIS characterization of the modified procedure of electrodes: (a) bare GCE, (b) GCE/AuNPs, (c) GCE/AuNPs/Antibodies, (d) GCE/AuNPs/Antibodies/BSA, and (e) GCE/AuNPs/Antibodies/BSA/Antigens.
Fig. 3. (A) Schematic illustration of the electrochemical immunoassay using different immunosensing probes: (a) PGN-Ab1/2, (b) Pt-Ab1/2, (c) labeled antibodies in 0.1 M PBS containing 4 mM GOD and HRP; Comparison of calibration curves of the electrochemical immunoassays for (B) CEA and (C) AFP. [Note: d is calibration curves for CEA and AFP in PBS which do not contain GOD and HRP.]
In addition, the present immunosensor exhibited a superior biosensing performance in PBS containing GOD and HRP (curve a) than that containing nothing (curve d). The presence of PGN not only obviously increased the surface area to capture a large amount of modified antibodies, and enzymes, but also accelerated the electron transfer. The immobilized GOD and HRP showed high catalytic efficiency with respect to glucose and H2O2. The result indicated that the immunosensor using PGN-Ab1/2 as signal probes exhibited high sensitivity as well as favored to detect CEA and AFP at very low concentrations. 3.7. Analytical performance of the multiplexed immunoassay To assess the performance of the proposed immunoassay, SWV measurements were carried out under the optimal experimental conditions after incubation with CEA and AFP standards and excess signal probes. As shown in Fig. 4A, the SWV reduction currents for THI at 1.5 V and for Fc at þ 3.5 V increased with an increase in the concentrations of CEA and AFP. The peak current of THI and Fc were separate proportional to the CEA (Fig. 4B) and AFP (Fig. 4C) concentrations in the working linear ranges of 0.01– 100 ng mL 1. The correlation coefficients were 0.998 and 0.994, respectively. The detection limits reached 1.64 pg mL 1 for CEA and 1.33 pg mL 1 for AFP at a signal-to-noise ratio of 3. The comparison of the working ranges and detection limits between the proposed biosensor and some recently published studies was listed in Table S2 (see Table S2 in the Supplementary information). The result indicated that the proposed electrochemical immunoassay using the triple signal amplification enabled relatively wide linear ranges and low LODs.
3.8. Analysis of clinical serum samples To investigate the analytical reliability and application potential of the proposed immunosensing method, ten human serum samples from Hospital of Capital Normal University (Beijing, China) were directly used for assay without dilution using the present method. The results using an enzyme-linked immunosorbent assay (ELISA) as a reference method were shown in Table S3 (see Table S3 in the Supplementary information). The relative errors between the two methods were both from 10% to 10% for CEA and AFP, indicating good accuracy of the proposed method for sample assay.
4. Conclusion In this work, we have successfully developed a novel ultrasensitive multiplexed electrochemical immunoassay for simultaneous detection of CEA and AFP using biofunctionalized graphene nanocomposites as distinguishable signal probes. The immunosensor with high sensitivity and wide linear ranges was achieved because of the performance of AuNPs and excellent electrocatalysis activities of the biofunctionalized graphene nanocomposites. Compared with other assays, highlights of this work can be summarized as follows: (1) A triple signal amplification strategy was constructed for ultrasensitive electrochemistry detection of CEA and AFP. (2) The immunosensor received a good correlation with ELISA as a reference. The multiple immunoassays with high selectivity, good stability and reproducibility could be readily applied in clinical diagnosis. (3) Such an immunosensor well
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Fig. 4. SWV responses (A) and calibration curves for different concentrations of CEA (B) and AFP (C) in PBS, pH 6.5, containing 4 mM GOD and HRP.
combines the simplicity of a single working-electrode platform and the amplified signal response without deoxygenation for simultaneous multianalyte determination in a single run. It can be foreseen that the universal method demonstrated here could be modified and extended to the detection of other multiple targets. Acknowledgments This research was financed by grants from the National Natural Science Foundation of China (21273153) and Beijing Natural Science Foundation (2132008).
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