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. 2022 Dec 23;12(1):22444.
doi: 10.1038/s41598-022-26768-w.

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

Jiaoling Huang  1 , Zhixun Xie  2 , Liji Xie  1 , Sisi Luo  1 , Tingting Zeng  1 , Yanfang Zhang  1 , Minxiu Zhang  1 , Sheng Wang  1 , Meng Li  1 , You Wei  1 , Qing Fan  1 , Zhiqin Xie  1 , Xianwen Deng  1 , Dan Li  1
Affiliations

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

Jiaoling Huang et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of the investigated nanocomposites. TEM images of G-Chi (a) and G-Chi-Au/PtNP (b). EDS elemental analysis of G-Chi-Au/PtNP (c).
Figure 2
Figure 2
(a) Nyquist plots and (b) semicircle diameters from EIS characterization of electrodes modified with different materials in 0.01 M PBS (pH = 7.0) containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl: (a-1) GE, (b-1) GE-G-Chi-PtNP, (c-1) GE-G-Chi-AuNP, (d-1) GE-G-Chi-Au/PtNP. Error bar = RSD ( n = 5).
Figure 3
Figure 3
Nyquist plots for EIS characterization of electrodes after different modification steps in electrolyte (pH = 7.0) containing 5 mM [Fe(CN)6]3−/4−: (a) after ARV/MAb immobilization on GE-G-Chi-Au/PtNP, (b) after blocking with BSA, (c) after incubation with 106.82 EID50 mL−1 ARV, (d) after blocking with BSA without ARV/MAb immobilization, (e) after incubation with 106.82 EID50 mL−1 ARV without ARV/MAb immobilization; (f) Ret from EIS characterization of electrodes at different modification steps in electrolyte (pH = 7.0) containing 5 mM [Fe(CN)6]3−/4−: (f-1) GE-G-Chi-Au/PtNP, (f-2) GE-G-Chi-Au/PtNP-Glu-ARV/MAb, (f-3) GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BAS, (f-4) GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BAS-ARV, (f-5) GE-G-Chi-Au/PtNP-Glu-BAS, (f-6) GE-G-Chi-Au/PtNP-Glu-BAS-ARV, (f-7) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb, (f-8) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA, (f-9) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA-ARV, (f-10) GE-G-Chi-Au/PtNP-EDC/NHS-BSA, (f-11) GE-G-Chi-Au/PtNP-EDC/NHS-BSA-ARV, (f-12) GE-G-Chi-Au/PtNP-ARV/MAb, (f-13) GE-G-Chi-Au/PtNP-ARV/MAb-BSA, (f-14) GE-G-Chi-Au/PtNP-ARV/MAb-BSA-ARV, (f-15) GE-G-Chi-Au/PtNP-BSA, (f-16) GE-G-Chi-Au/PtNP-BSA-ARV, (f-17) GE-G-Chi-Au/PtNP-CH-ARV/MAb, (f-18) GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA, (f-19) GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA-ARV, (f-20) GE-G-Chi-Au/PtNP-CH-BSA, (f-21) GE-G-Chi-Au/PtNP-CH-BSA-ARV. Error bar = RSD (n = 5).
Figure 4
Figure 4
(a) Nyquist plots for different concentrations of ARV on GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA; (b) Calibration curve of GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA with different concentrations of ARV. Error bar = RSD (n = 5). (c) Nyquist plots for different concentrations of ARV on GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA; (d) Calibration curve of GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA with different concentrations of ARV. Error bar = RSD (n = 5). (e) Nyquist plots for different concentrations of ARV on GE-G-Chi-Au/PtNP-ARV/MAb-BSA; (f) Calibration curve for GE-G-Chi-Au/PtNP-ARV/MAb-BSA with different concentrations of ARV. Error bar = RSD (n = 5). (g) Nyquist plots for different concentrations of ARV on GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA; (h) Calibration curve for GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA with different concentrations of ARV. Error bar = RSD (n = 5). (a-1) 0, (b-1) 100.82 EID50 mL−1 ARV, (c-1) 101.82 EID50 mL−1 ARV, (d-1) 102.82 EID50 mL−1 ARV, (e-1) 103.82 EID50 mL−1 ARV, (f-1) 104.82 EID50 mL−1 ARV, (g-1) 105.82 EID50 mL−1 ARV, (f-1) 106.82 EID50 mL−1 ARV.
Figure 5
Figure 5
Schematic illustration of the electrochemical immunosensors.
Figure 6
Figure 6
Specificity of the immunosensors toward the target (ARV) and other interfering substances. (a) GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA, (b) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA, (c) GE-G-Chi-Au/PtNP-ARV/MAb-BSA, (d) GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA. Error bar = RSD (n = 5).
Figure 7
Figure 7
Reproducibility of the four immunosensors in the presence of 103.82 EID50 mL−1 ARV (a) GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA, (b) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA, (c) GE-G-Chi-Au/PtNP-ARV/MAb-BSA, (d) GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA. Error bar = RSD (n = 5).
Figure 8
Figure 8
Long-term stability of the proposed immunosensors: (a) GE-G-Chi-Au/PtNP-Glu-ARV/MAb-BSA, (b) GE-G-Chi-Au/PtNP-EDC/NHS-ARV/MAb-BSA, (c) GE-G-Chi-Au/PtNP-ARV/MAb-BSA, (d) GE-G-Chi-Au/PtNP-CH-ARV/MAb-BSA.
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