Ultrasensitive and label free electrochemical immunosensor for detection of ROR1 as an oncofetal biomarker using gold nanoparticles assisted LDH/rGO nanocomposite

Abstract

In the present article, we developed a highly sensitive label-free electrochemical immunosensor based on NiFe-layered double hydroxides (LDH)/reduced graphene oxide (rGO)/gold nanoparticles modified glassy carbon electrode for the determination of receptor tyrosine kinase-like orphan receptor (ROR)-1. In this electrochemical immunoassay platform, NiFe-LDH/rGO was used due to great electron mobility, high specific surface area and flexible structures, while Au nanoparticles were prepared and coated on the modified electrodes to improve the detection sensitivity and ROR1 antibody immobilizing (ROR1Ab). The modification procedure was approved by using cyclic voltammetry and differential pulse voltammetry based on the response of peak current to the step by step modifications. Under optimum conditions, the experimental results showed that the immunosensor revealed a sensitive response to ROR1 in the range of 0.01-1 pg mL^-1, and with a lower limit of quantification of 10 attogram/mL (10 ag mL^-1). Furthermore, the designed immunosensor was applied for the analysis of ROR1 in several serum samples of chronic lymphocytic leukemia suffering patients with acceptable results, and it also exhibited good selectivity, reproducibility and stability.

Scheme 1

Schematically presentation of Electrode preparation steps.

Figure 1

(A) The differential pulse voltammetry (DPV) of immobilization steps of the modified electrode in Fe(CN)63−/4−. Scan rate, 100 mV/s; (a) bare GCE, (b) NiFe-LDH/rGO, (c) NiFe-LDH/rGO/AuNPs, (d) NiFe-LDH/rGO/AuNPs/ROR1sAb, (e) NiFe-LDH/rGO/AuNPs/ROR1sAb/BSA, and (f) NiFe-LDH/rGO/AuNPs/ROR1sAb/BSA/ROR1sAg. (B) The cyclic voltammetry (CV) of immobilization steps of the modified electrode in Fe(CN)63−/4−. Scan rate, 100 mV/s.

Figure 2

SEM images of NiFe-LDH/rGO nanocomposites (A); NiFe-LDH/rGO/AuNPs nanocomposites (B); EDS spectra of NiFe-LDH/rGO (C) and NiFe-LDH/rGO/AuNPs (D) on the GCE.

Figure 3

Optimization of the experimental conditions. Effect of (A) NiFe-LDH/rGO electrodeposition cycles number; (B) the AuNPs electrodeposition cycles number and (C) different anti-ROR1 concentration on the current response.

Figure 4

(A) DPV curves of the immunosensor after incubation with different concentrations of ROR1 (0.01 fg mL⁻¹, 0.1 fg mL⁻¹, 1 fg mL⁻¹, 0.01 pg mL⁻¹, 0.1 pg mL⁻¹, 1 pg mL⁻¹). (B) The linear relationship between the peak current versus the logarithm concentration of ROR1 in the range of 0.01 fg mL⁻¹ to 1 pg mL⁻¹. Error bars represent standard deviation (n = 3).

Figure 5

(A) Selectivity of immunosensor to ROR1 (1 pg mL⁻¹) compared with four nonspecific interferences including cMet (1 pg ml⁻¹), fzd7 (1 pg ml⁻¹) and BSA (1 pg ml⁻¹) (n = 3). (B) reproducibility of the immunosensor measured by analyzing the ROR1 with concentration of 1 fg ml⁻¹ with five equal electrodes prepared by the same method. (C) stability of the ROR1 immunosensor kept at 7 and 15 days in refrigerator at 4 °C.

Figure 6

Real sample analysis of ROR1 biomarker in five clinical CLL human serum samples.