key: cord-0835527-xh5u7q7c
authors: Shu, Jiangnan; Shen, Wen; Cui, Hua
title: Ultrasensitive label-free electrochemiluminescence immunosensor based on N-(4-aminobutyl)-N-ethylisoluminol-functionalized graphene composite
date: 2015-01-22
journal: Sci China Chem
DOI: 10.1007/s11426-015-5320-2
sha: 6f396dca9b8d5d8e40af2f4aab4c91584ecd2776
doc_id: 835527
cord_uid: xh5u7q7c

The electrochemiluminescence (ECL) behavior of N-(4-aminobutyl)-N-ethylisoluminol (ABEI)-functionalized graphene composite (ABEI-GC) modified on an indium tin oxide (ITO) electrode was studied. ABEI-GC exhibited excellent ECL activity. On this basis, a label-free ECL immunosensor was developed for the sensitive detection of human immunoglobulin G (hIgG) by using ABEI-GC as the ECL nano-interface via a layer-by-layer assembly technique. ABEI-GC was first assembled onto an ITO electrode. Positively charged chitosan was then electrostatically adsorbed to the modified electrode. Finally, negatively charged antibody-coated gold nanoparticles were attached to the surface to form the ECL immunosensor. In the presence of hIgG, hIgG was captured by its antibody. In addition, an ECL signal was detected in the presence of H(2)O(2) when a double potential was applied. The ECL immunosensor for the determination of hIgG showed a linear range of 1.0×10(−13)–1.0×10(−8) g/mL with a detection limit of 5.0×10(−14) g/mL. This immunosensor has high sensitivity, wide linearity and good reproducibility. The superior sensitivity of the proposed ECL immunoassay mainly derives from the incorporation of ABEI-GC, which not only improves the ECL intensity, response speed, and stability, but also provides a large specific surface for high levels of protein loading. This work reveals that ABEI-GC is good nano-interface for the construction of ECL biosensors. Our strategy is promising for protein detection and may open up a new avenue for ultrasensitive label-free immunoassays.

Immunosensors have attracted considerable attention so as to obtain quick and sensitive immunological responses for environmental monitoring, food safety, and clinical diagnoses [1,2]. However, conventional immunoassays based on labeling techniques are generally tedious, time-consuming, and costly, and may lead to the denaturizing of the modified biomolecules [3] [4] [5] . Therefore, there has been great interest in developing low-cost label-free biosensors. The main advantage of label-free strategies is that they do not require the labeling of the antibody or antigen with markers, which makes the experimental process simple, low cost, and timesaving. Label-free immunoassays combined with various detection techniques such as piezoelectricity [6] , surface plasmon resonance [7] , electrochemistry [8] , chemiluminescence (CL) [9] , and ECL [10, 11] have been developed.

As a valuable detection method, ECL has been widely applied to clinical diagnosis, environment monitoring, food safety, and pharmaceutical analysis due to acknowledged advantages such as versatility, simplified optical setup, very low background signal, and good temporal and spatial control [12] [13] [14] [15] [16] . Recently, label-free ECL immunoassays have also been reported for the determination of small molecules and biomacromolecules and have shown high sensitivity, specificity, rapidity, simplicity, and low cost [11, 17] .

Recently, graphene has attracted considerable attention due to its high electrical conductivity, high surface-tovolume ratio, high electron-transfer rate, and exceptional thermal stability [18, 19] . To expand the use of graphene in various applications, much effort has been made in terms of its functionalization so as to obtain advanced functional composites with superior characteristics [20] [21] [22] . In our previous work, graphene was successfully functionalized with ABEI via - interactions that exhibited good CL activity [23] . In view of the high electrical conductivity and electrocatalytic activity of graphene and the good ECL activity of ABEI, such composites might also possess good ECL characteristics. In addition, graphene has the advantages of high surface-to-volume ratio and good biocompatibility, which are beneficial to the construction of biosensing platforms. Accordingly, it provides a strong possibility for developing an ultrasensitive label-free ECL immunosensor based on ABEI-GC as the nano-interface.

We studied the ECL behavior of an ABEI-GC-modified ITO electrode. The ABEI-GC-modified ITO electrode in alkaline solution exhibited good ECL activity in the presence of H 2 O 2 . On this basis, we proposed a label-free ECL immunosensor by using ABEI-GC as the nano-interface via a layer-by-layer assembly method that takes hIgG as a model target. The fabrication of the immunosensor was studied by electrochemical impedance spectroscopy (EIS). The conditions for ECL detection were optimized. The analytical performance and the specificity of the immunosensor were examined. The applicability of the proposed immunosensor in real human-serum samples was investigated.

ABEI was purchased from Sigma-Aldrich (USA). Graphene oxide (GO) was purchased from XFNANO Materials Tech Co. Ltd. (Nanjing, China). Bovine serum albumins (BSA), hIgG, and goat-antihuman IgG were obtained from Solarbio (Beijing, China). Chloroauric acid (HAuCl 4 ·4H 2 O) was obtained from Shanghai Reagent Company (Shanghai, China). A HAuCl 4 stock solution (2% HAuCl 4 , w/w) was prepared by dissolving 1.0 g of HAuCl 4 ·4H 2 O in 412 mL of purified water and storing the mixture at 4 °C. Chitosan (95% deacetylation) was prepared by dissolving chitosan in 0.1 mol/L acetic acid solution, filtering the insoluble substance, and adjusting the pH to 5.0 with 0.1 mol/L sodium hydroxide solution. The buffer solutions used in the experiment included carbonate buffer (CBS, NaHCO 3 -Na 2 CO 3 ) and phosphate saline buffer (PBS). All of the other reagents were of analytical grade. Ultrapure water was prepared by a Milli-Q system (Milli-pore, France) and used throughout.

Electrochemical and ECL measurements were performed with a homemade ECL/Electrochemical cell system, including a model CHI 760B electrochemistry work-station (Chenhua, China), an H-type electrochemical cell (homemade) [24] , a model CR-105 photomultiplier tube (PMT) (Bingsong, China) and a model RFL-1 luminometer (Ruimai, China). The EIS experiment was performed with a CHI 760B electrochemistry workstation (Chenhua, China). A PST-60 HL plus Thermo Shaker (Biosan, Latvia) was used to control the temperature of the reactions.

The ABEI-GC was synthesized as described previously [23] . First, 5 mL (2 mg/mL) of aqueous ABEI alkaline solution was added to 5 mL (0.2 mg/mL) of exfoliated GO suspension. After ultrasonication for 30 min, the mixture was vigorously stirred at 80 °C for 24 h. Next, the dialysis procedure was used to remove the free ABEI and the coexisting free molecules from the ABEI-GC dispersion.

Gold nanoparticles (AuNPs) were prepared by the trisodium citrate reduction method according to a previous study, with modification [25] . Briefly, 5.0 mL of 1% trisodium citrate solution was rapidly added to 100 mL 0.3 mmol/L boiling HAuCl 4 solution with fierce stirring. The mixture was refluxed for 30 min and then naturally cooled to room temperature. After being filtered through a 0.22 m cellulose membrane, the AuNPs solution was adjusted to pH 8.0 with diluted NaOH solution. The goat-antihuman IgG functionalized AuNPs (Ab-AuNPs) were prepared according to previously reported studies, with modification via electrostatic interaction as well as hydrophobic interaction and weak covalent interactions [5, 26] . Next, 25 L of 1.0 mg/mL goat-antihuman IgG antibody was added to 1.0 mL of the pH 8.0 AuNPs solution, followed by incubation at room temperature for 30 min. The solution was added, with 5% BSA solution, to the final concentration of 1% BSA and then stirred for 5 min. The conjugate was centrifuged at 17120 r/min for 40 min (Universal 320, Hettich, Germany) to remove the excess reagent, and the soft sediment was dissolved in PBS buffer (0.1 mol/L, pH 7.4) containing 1% (w/w) BSA to obtain the Ab-AuNPs. A CD experiment demonstrated that the Ab was successfully conjugated to the AuNPs.

ITO-coated glass slides were used as assembly electrodes owing to their stability, easy miniaturization, and compatibility with microfabrication [27] . ITO-coated glass slides with dimensions of 6.0 cm×1.5 cm were cleaned by sequential sonication in neat acetone, ethanol, and ultrapure water (15 min each). The ITO substrate was reversibly bound to a punched poly(dimethylsiloxane) (PDMS) layer that was prepared by curing the mixture of PDMS monomer and the curing agent (10:1) in an oven at 75 °C for 1.5 h. The effective electrode area was confined by a punched round hole of 5 mm in diamter, which was used as a reservoir for subsequent assembly.

The ECL immunosensor was assembled via the layerby-layer method ( Figure 1 ). First, 50 L ABEI-GC solution was dropped into the reservoir and dried naturally in air. Then, 10 L 5% chitosan solution was dropped onto the ABEI-GC modified electrode and dried in air. Later, the modified electrode was rinsed with ultrapure water to remove the physically adsorbed chitosan. Subsequently, 50 L Ab-AuNPs solution was added to the reservoir and incubated at 4 °C for 8 h and rinsed again with ultrapure water. Next, 50 L 1% (w/w) BSA in 0.01 mol/L PBS (pH 7.4) was used for blocking non-specific binding sites for 30 min at 37 °C, followed by a thorough washing with 0.02 mol/L PBS. Finally, 50 L of different concentrations of hIgG were dropped on the modified electrode at 37 °C for 40 min, followed by a thorough washing with 0.02 mol/L PBS. In this way, the hIgG/BSA/Ab-AuNPs/CS/ABEI-GC/ITOmodified ITO substrate (i.e., the immunosensor) was successfully constructed.

ECL measurements were carried out on a three-electrode homemade ECL system. The modified ITO electrode described above served as the working electrode; a platinum wire served as the counter-electrode; and a silver wire served as the quasireference electrode (AgQRE). We chose to use an AgQRE due to the simplicity of its cell construction and quick potential response. Although the potential of the AgQRE was found to be essentially stable during an experiment, measurements of ∆E=E Ag/Ag+ E SCE in different solutions were taken for potential calibrations. During the ECL measurements, a portion of the CBS buffer (0.02 mol/L, pH 9.95) containing 1 mmol/L H 2 O 2 was transferred into the working compartment and the auxiliary compartment of the ECL cell. When a double-step potential (30 s pulse period, 0.1 s pulse time, 0.6 V pulse potential, and 0 V initial potential) was applied to the working electrode, the ECL signal was generated and recorded.

The electrochemical and ECL behaviors of ABEI-GCmodified ITO electrode were studied. Cyclic voltammograms (CVs) were taken from 0 to 1.0 V in 0.02 mol/L CBS (pH 9.95) containing 1 mmol/L H 2 O 2 . In Figure 2 , Curves a and b respectively depict the curves of ECL intensity versus the potential and CV of the ABEI-GC-modified ITO electrode. The ABEI-GC modified ITO electrode exhibited good ECL activity. The ECL emission started at 0.25 V and the ECL peak value was achieved at around 0.7 V; the latter was related to electro-oxidation of ABEI on the ITO electrode according to the CV of pure ABEI (Figure 2 , Curve c). These results indicated that the ECL emission was from ABEI molecules attached on the surface of graphene. Therefore, it is feasible to fabricate a label-free ECL immunosensor based on such an ECL functionalized nanointerface.

We also studied the ECL behavior of ABEI-GC under a double-step potential on the ABEI-GC-modified ITO electrode in 0.02 mol/L CBS (pH 9.95) containing 1 mmol/L H 2 O 2 (Figure 3 ). When the double-step potential was applied to the electrode, a pulse ECL signal was obtained. Figure 3 displays the ECL signals for 10 periods, which were much better than those obtained by CV in both signal intensity and stability. Therefore, double-step potential was chosen for the immunosensor in the following experiments. Figure 1 displays the procedure and the working prinicple for fabricating a label-free ECL immunosensor for human immunoglobulin detection. First, ABEI-GC was selfassembled on the surface of the ITO substance through physical absorption, to provide ECL-functionalized nanointerface. Next, positively charged chitosan was adsorbed to the nano-interface of the ABEI-GC through electrostatic interaction. After that, negatively charged Ab-AuNPs were further assembled on the ABEI-GC-modified electrode via electrostatic interaction. Next, BSA was used for blocking the non-specific binding sites. Finally, hIgG was attached on the modified electrode via specific binding between goat-antihuman IgG and hIgG.

EIS was used to monitor the process of electrode modification ( Figure 4 ). The results were consistent with the fact that the electrode was modified as expected. The impedance spectra consisted of a semicircle at high ac modulation frequency and a line at low ac modulation frequency, which demonstrated that the electrode process was controlled by electron transfer at high frequency and by diffusion at low frequency. The electron transfer resistance decreased with the decrease of the diameter of the semicircle. Our stepwise EIS measurements of the modified ITO electrode at different modification steps were carried out at the PDMS reservoir because this placement ensured the same electrode area. It can be seen that when ABEI-GC was assembled on the ITO electrode, the electron transfer resistance increased remarkably (Curve b) compared with the bare ITO electrode (Curve a). After further modification with chitosan, an insulating layer was created on the surface of as-prepared electrode, resulting in an increased electron transfer resistance (Curve c). Once the Ab-AuNPs were further assembled on the electrode (Curve d), the electron transfer resistance decreased significantly due to the superior conductivity of AuNPs, which facilitated the electron transfer between the protein active center and the electrode surface and led to a net decrease in electron transfer resistance. After further incorporation of BSA, the electron-transfer resistance elevated largely as expected (Curve e), because the proteins blocked the electron transfer. Therefore, we increased the electron transfer resistance after more hIgG was attached to the surface of the modified electrode (Curve f). The entire EIS dataset indicated that the electrode was modified as expected.

The process of electrode modification was also investigated by the ECL behavior of each step. The ECL responses of the modified electrodes at different stages were examined as shown in Figure 5 . Strong ECL signals were observed after the ABEI-GC was assembled on the ITO electrode (Curve a), because of the good ECL property of ABEI-GC. When assembled with chitosan, the ECL intensity decreased dramatically, owing to the insulating property of chitosan (Curve b). The ECL intensity increased significantly after Ab-AuNPs were assembled on the electrode (Curve c), which contributed to the superior conductivity and the catalytic characteristics of AuNPs. After bounding with BSA, the ECL intensity was much reduced because the proteins blocked the electron transfer (Curve d). Similarly, the ECL intensity decreased after further incorporation of hIgG (Curve e). Accordingly, we might quantitatively evaluate the concentration of hIgG according to ECL response.

A double-step potential was applied to the electrode to initi-ate the ECL signal of the immunosensor in the presence of H 2 O 2 (Figure 1) . To obtain optimal ECL signaling, the effects of pH value, pulse period, pulse time, pulse potential, initial potential, and concentration of H 2 O 2 on the ECL intensity should be investigated. The effect of pH on the ECL intensity was examined in the range of 8.95-11.37 in 0.02 mol/L CBS. Figure 6(a) shows the pH dependence of the ECL intensity; maximal ECL intensity was obtained at pH 9.95. The effect of the initial pulse potential in the range of 0.3-0.3 V was also investigated (Figure 6(b) ). When the initial pulse potential was 0 V, maximal ECL intensity was achieved; probably because the initial pulse potential of 0 V had a better diffusion controlled reaction on the surface of the modified electrode. Therefore, an initial pulse potential of 0 V was used in the following experiments. The effect of the pulse potential in the range of 0.4-0.8 V was studied as well ( Figure 6(c) ). The ECL intensity increased with the increase of the pulse potential because the electro-oxidation was much faster at higher electrode potential. A pulse potential of 0.6 V was adopted because the stability and reproducibility of the modified electrode declined if a high potential (above 0.6 V) was used. The effect of pulse time on the ECL intensity was examined in the range of 0.025-0.125 s (Figure 6(d) ). The ECL intensity increased with the pulse time. However, when the pulse time was longer than 0.1 s, the ECL intensity decreased rapidly at the second period and could not reach a stable ECL signal in the following period. This result occurred because the diffusion layer on the surface of electrode became thicker in a relatively long pulse time and could not recover in the next pulse. Therefore, a pulse time of 0.1 s was selected. The effect of the pulse period on the ECL intensity was investigated in the range of 10-50 s (Figure 6(e) ). The ECL intensity increased with the pulse period, which might have been due to more-effective diffusion of H 2 O 2 in a longer pulse period. To obtain a shorter analytical time and higher ECL intensity, a pulse period of 30 s was adopted in the following experiments. The ECL intensity increased with the increase of the concentration of H 2 O 2 and reached maximum at 1.0 mmol/L ( Figure 6 (f)). Therefore, 1.0 mmol/L H 2 O 2 was used in the following experiments.

Under the optimized conditions, label-free ECL immunosensor was employed for the detection of hIgG. The ECL signal increased with the increment of hIgG concentration (Figure 7) , which exhibited a wide linear dynamic range of 1.0×10 13 -1.0×10 8 g/mL. The regression equation was I=3417.1529.3logC and the correlation coefficient was 0.996, where I was the ECL intensity and C was the concentration of hIgG (g/mL). The limit of detection for hIgG at a signal-to-noise ratio of 3 (S/N=3) was 5.0×10 14 g/mL. The precision of the ECL immunosensor was also investi- gated. The relative standard deviations (RSD) of five replicate determinations of hIgG within a day (intra-day precision, n=5) was 3.27%; the RSD in 7 days (inter-day precision, n=7) was 8.03%. These results indicated acceptable reproducibility.

To affirm the specificity of the label-free ECL immunosensor, BSA, lysozyme, hemoglobin (HGB), and human serum albumin (HSA) with 1.0×10 9 g/mL concentration were chosen as interferents. They were used for immunoassays instead of hIgG. As can be seen in Figure 8 , the ECL response from BSA, lysozyme, HGB, and HSA with concentrations of 10× over hIgG were comparable with the blank. A much weaker ECL response was observed when 1.0×10 10 g/mL hIgG was used for the ECL immunoassay. These results demonstrate that the developed label-free ECL immunosensor is selective for the detection of hIgG.

A comparison of the proposed method with other labelfree immunoassays for the determination of hIgG was made (Table 1) [1, [28] [29] [30] [31] [32] [33] . The results show that the limit of detection of the present method is superior to most previously reported methods for the determination of hIgG. Although the present label-free ECL immunosensor exhibited a higher detection limit than the assays based on graphene-quantum dots composites amplification via the conjugation of PDDA and GNPs, it avoided complicated experimental procedures. This avoidance made the method low cost, easy to operate, and time-saving. Moreover, the present method showed good precision as well as acceptable stability and reproducibility. Therefore, the proposed method is promising for practical applications in fields such as clinical diagnosis, nano-Au-ERGO AuNPs ECL 1.3 [32] P-GR-CdSe PDDA/AuNPs ECL 0.005 [33] environment monitoring, food safety, pharmaceutical analysis, etc. 

The clinical applicability of the proposed method was investigated by analyzing several real samples of human serum. Before the test, the samples were appropriately diluted step by step to be in the linear range of the proposed method. As shown in Table 2 , the recoveries were in the range of 97%-104%, which indicated that the developed label-free ECL immunoassay could be applied for the determination of hIgG in clinical diagnosis.

An advanced label-free ECL immunosensor is proposed for the sensitive detection of hIgG by using ABEI-GC to build a CL-sensing platform. Due to the high conductivity and the electrocatalytic activity of the graphene-based immunosensor as well as the superior ECL property of ABEI-GC, trace amounts of hIgG were successfully determined by the as-prepared immunosensor with good ECL response. Furthermore, the large specific surface area of graphene afforded high protein loading and the gold nanoparticles could further enhance the ECL intensity. As a result, the sensor showed an extremely sensitive response to hIgG in a linear range of 1.0×10 13 -1.0×10 8 g/mL with a detection limit of 5.0×10 14 g/mL. Moreover, the label-free ECL immunosensor showed good precision as well as acceptable stability and reproducibility, which indicate great potential in clinical applications. This research provides a promising technique for protein detection, which may open up a new avenue for ultrasensitive label-free immunoassays. Encapsulation of biomolecules for bioanalytical purposes: preparation of diclofenac antibody-doped nanometer-sized silica particles by reverse micelle and sol-gel processing. Anal Chim Acta, 2006, 573:

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