Explore how immobilization strategies affected immunosensor performance by comparing four methods for antibody immobilization on electrode surfaces

Among the common methods used for antibody immobilization on electrode surfaces, which is the best available option for immunosensor fabrication? To answer this question, we first used graphene-chitosan-Au/Pt nanoparticle (G-Chi-Au/PtNP) nanocomposites to modify a gold electrode (GE). Second, avian reovirus monoclonal antibody (ARV/MAb) was immobilized on the GE surface by using four common methods, which included glutaraldehyde (Glu), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS), direct incubation or cysteamine hydrochloride (CH). Third, the electrodes were incubated with bovine serum albumin, four different avian reovirus (ARV) immunosensors were obtained. Last, the four ARV immunosensors were used to detect ARV. The results showed that the ARV immunosensors immobilized via Glu, EDC/NHS, direct incubation or CH showed detection limits of 100.63 EID50 mL−1, 100.48 EID50 mL−1, 100.37 EID50 mL−1 and 100.46 EID50 mL−1 ARV (S/N = 3) and quantification limits of 101.15 EID50 mL−1, and 101.00 EID50 mL−1, 100.89 EID50 mL−1 and 100.98 EID50 mL−1 ARV (S/N = 10), respectively, while the linear range of the immunosensor immobilized via CH (0–105.82 EID50 mL−1 ARV) was 10 times broader than that of the immunosensor immobilized via direct incubation (0–104.82 EID50 mL−1 ARV) and 100 times broader than those of the immunosensors immobilized via Glu (0–103.82 EID50 mL−1 ARV) or EDC/NHS (0–103.82 EID50 mL−1 ARV). And the four immunosensors showed excellent selectivity, reproducibility and stability.

www.nature.com/scientificreports/ potential for application in electrochemical immunosensors because of its low manufacturing cost, superior conductivity and large specific surface area 13 . Among metal nanomaterials, gold nanoparticles (AuNPs) and platinum nanoparticles (PtNPs) are most widely used in electrochemical immunosensors because of their excellent performance and excellent conductivity 14,15 . Additionally, G has been functionalized and adsorbed on chitosan (Chi) through π-π stacking to form graphene-chitosan (G-Chi) hybrid materials 16 . In the G-Chi hybrid materials, Chi chelates Au 3+ and Pt 2+ metal ions and acts as a reducing agent to convert the Au 3+ and Pt 2+ ions into AuNPs and PtNPs 17,18 ; further, G-Chi hybrid materials can be loaded with substantial amounts of AuNPs and PtNPs because of the large specific surface area of G. Hence, we designed a "label-free" electrochemical immunosensor based on G-Chi-Au/PtNP nanocomposites in this work. In addition, effective immobilization of antibodies is an essential step in constructing electrochemical immunosensors and constitute another important factor in improving the performance of the electrochemical immunosensors 19 . The sensitivities and linear ranges of electrochemical immunosensors are limited by the antibody immobilization strategy chosen 20 . Various antibody immobilization strategies, including glutaraldehyde (Glu) cross-linking, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry, direct incubation and cysteamine hydrochloride (CH), have been exploited by different research groups [21][22][23][24][25][26] , but it is not known which approach is best. To answer this question, four immobilization strategies were compared in the present study: (1) Glu immobilization, (2) EDC/NHS immobilization, (3) direct immobilization and (4) CH immobilization. The results showed that the linear range obtained with the CH immobilization strategy was 10 times broader than that realized with the direct immobilization strategy and 100 times broader than those seen with the Glu immobilization strategy and EDC/NHS immobilization strategy, and their detection limits were similar.

Results and discussion
Nanoparticle synthesis and characterization. A transmission electron microscopy (TEM) micrograph of G-Chi, which has a thin, wrinkled, rippled and flake-like structure, is shown in Fig. 1a. Figure 1b shows the TEM micrograph of G-Chi-Au/PtNP, which indicates that Au/Pt was loaded on the surface of G-Chi. In addition, energy dispersive spectroscopy (EDS) elemental analysis of G-Chi-Au/PtNP was employed to determine the presence of Au and Pt, which confirmed that Au/PtNP had been loaded on the surface of G-Chi (Fig. 1c). The mechanism for formation of G-Chi-Au/PtNP involved Au 3+ and Pt 2+ adsorption from aqueous solution due to chelation by G-Chi and then reduction to Au/Pt nanoparticles by Chi. Chi was used as both a stabilizing agent and reducing agent.
Electrochemical characterization. Electrochemical impedance spectroscopy (EIS) is an effective technique for probing the features of surface modified electrodes, and it sensitively analyzes the interactions of analytes with modified electrodes and produces measurable electric signals. More important, EIS is more sensitive than either amperometric or voltammetric methods 27,28 . In typical EIS Nyquist plots, a semicircle appears in the high-frequency region, while a line appears in the low-frequency region, and the diameter of the semicircle corresponds to the electron transfer resistance (R et ). In brief, the resistance on the surface of the electrode can be estimated by determining the semicircle diameter. Here, EIS was employed to characterize which material is better for electrode modification, and the results are shown in Fig. 2. The diameter of the semicircle in the Nyquist plot of GE corresponds to an impedance of 1561 Ω, which was decreased to 1167 Ω, 950 Ω and 439 Ω upon modification of the GE with G-Chi-PtNP, G-Chi-AuNP, and G-Chi-Au/PtNP, respectively, due to the high conductivities of G, AuNP and PtNP. These results showed that G-Chi-Au/PtNP exhibited the fastest electron transfer, and it was selected as the material for GE modification.
In addition, EIS was employed to survey the layer-by-layer modification of GE. Figure  MAb-BSA were incubated with 10 6.82 EID 50 mL −1 ARV, the ARV was adsorbed on the electrodes via a specific response with ARV/MAb, and the corresponding R et s were further increased to 4841 Ω, 7468 Ω, 5547 Ω, and 9417 Ω (Fig. 3c,f) because electron transfer to the surface of the electrode was impeded by the ARV protein, and the results demonstrated that the ability of the four different immunosensors to combine ARV decreased in the order CH > direct incubation > EDC/NHS > Glu. Most importantly, the different relative orders for ARV/ MAb immobilization and the ARV immobilization demonstrated that the number of ARVs adsorbed on the immunosensors was not only related to the number of ARVs/MAbs but was also affected by the method used to immobilize the ARVs/MAbs.
In addition, BSA was immobilized on GE-G-Chi-Au/PtNP by four different methods to evaluate the ability of the four methods to immobilize protein again, the results are shown in Fig. 3d (Fig. 4g,h). These results show that the sensitivities of the four immunosensors were slightly different, while saturation beyond the extended dynamic range of GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA was 10 times that of GE-G-Chi-Au/PtNP-ARV/MAb-BSA and 100 times those of GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA and GE-G-Chi-Au/ PtNP-EDC/NHS-ARV/MAb-BSA. These results were attributed to the different methods of ARV/MAb immobilization. ARV/MAb is a Y-shaped protein that consists of two light and two heavy chains linked by disulfide bonds, and ARV/MAb is monomeric (IgG) and contains the F ab region and F c region (Fig. 5). The F ab region, which participates in antigen binding, consists of an amino end, while the F c region, which is the stem of the Y shape, has a carboxyl end group 29,30 . The region (F ab or F c ) of ARV/MAb attached to the electrode depended on immobilization strategy used. For GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA, Glu was used as the coupling agent to connect amine groups on the surface of the electrode and amine groups (F ab region) on ARV/MAb. The EDC/ NHS-based immobilization strategy for GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA activated carboxyl groups on the surface of the electrode and allowed NHS ester groups to act as intermediates leading to covalent attachment of the activated carboxyl groups with amino groups on the surface of the electrode present in the F ab region on ARV/MAb. The antibodies in GE-G-Chi-Au/PtNP-ARV/MAb-BSA and GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA were immobilized onto the electrode via the tail end of the F c region through covalent attachments between electrode surface amino groups and carboxyl groups on ARV/MAb, which resulted in accessibility for antigen binding because it was located in an orthogonal position. However, attachment of ARV/MAb onto

Conclusions
In this work, four different antibody immobilization strategies (Glu as a coupling agent to connect amine groups on the surface of the electrode and amine groups present in ARV/Mab; EDC/NHS chemistry to activate carboxyl groups on the surface of the electrode for covalent attachment with amine groups present at the F ab region on ARV/Mab; immobilization of ARV/MAb directly onto the amine group ending the electrode; and immobilization   Second, G-Chi was prepared according to our previous report 33 . Briefly, 0.05 mg Chi powder was added to 100 mL 1.0% (v/v) acetic acid solution under continuous stirring at room temperature and maintained for 0.5 h. Then, 100 mg of G was added, the mixture was continuously stirred for 24 h, and G-Chi was collected by centrifugation and washed with dd-H 2 O.

Synthesis of G-Chi-Au
Third, 1 mL 10 mmol/L HAuCl 4 , 1 mL 10 mmol/L K 2 PtCl 4 and 20 mL 1 mg/mL G-Chi solution were mixed together, and the mixture was stirred at room temperature for 3 h. Then, the mixture was heated to 80 °C under continuous stirring for 1 h to obtain the G-Chi-Au/PtNP nanocomposite.
Fabrication of the electrochemical immunosensor. The step-by-step fabrication of the electrochemical immunosensor is illustrated in Fig. 5. A gold electrode (GE) was polished with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powders, rinsed with ddH 2 O, and cleaned by sonication in ddH 2 O, ethanol, and ddH 2 O for 5 min each. Subsequently, the GE was dried by flushing with nitrogen gas.
Next, 8 μL of prepared G-Chi-Au/PtNP was dropped onto the surface of the clean GE and dried at room temperature, and G-Chi-Au/PtNP-GE was obtained. ARV/MAbs was immobilized onto G-Chi-Au/PtNP-GE by four different strategies: (1) G-Chi-Au/PtNP-GE was incubated with 10 μL of 5% Glu for 3 h and washed with PBS three times, and then 8 μL of 100 μg/mL ARV/MAbs was deposited onto G-Chi-Au/PtNP-Glu-GE and incubated at 4 °C for 8 h; (2) G-Chi-Au/PtNP-GE was anodized in 0.5 mol/L NaOH solution with a potential of + 1.3 V for 40 s to increase the number of -COOH groups on its surface. The anodized G-Chi-Au/PtNP-GE was incubated with 10 μL of solution that contained 50 mmol/L EDC and 30 mmol/L NHS in MES (pH 4.7) at room temperature for 1 h. -COOH groups were converted to amine-reactive NHS esters in this step to prepare for ARV/mAb immobilization. Then, after washing with PBS (pH 7.4) three times to remove the unreacted EDC/NHS, 8 μL 100 μg/mL ARV/MAbs was deposited onto the NHS-activated surface of G-Chi-Au/PtNP-EDC/NHS-GE, which was then incubated at 4 °C for 8 h; (3) 8 μL 100 μg/mL ARV/MAbs was deposited onto G-Chi-Au/PtNP-GE and incubated at 4 °C for 8 h without any further modification; and (4) G-Chi-Au/PtNP-GE was incubated with 10 μL 2 mg/mL CH at room temperature in the dark for 4 h and washed with PBS three times, after which 8 μL 100 μg/mL ARV/MAbs was deposited onto G-Chi-Au/PtNP-CH-GE, and the material was incubated at 4 °C for 8 h. All the electrodes with immobilized ARV/MAbs prepared via above four strategies were washed with PBS (pH 7.4) to remove physically adsorbed or excess ARV/MAbs, incubated with 1% BSA in 0.01 mol/L PBS (pH 7.4) at room temperature for 1 h to block the free active sites on the electrodes and washed three time with PBS (pH 7.4). The obtained immunosensors were denoted GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA, GE-G-Chi-Au/