An ultra-sensitive label-free electrochemiluminescence CKMB immunosensor using a novel nanocomposite-modified printed electrode

Abstract

This study presents a novel and ultrasensitive electrochemiluminescence approach for the quantitative assessment of creatine kinase MB (CK-MB). Both carbon, carbon nano-onions (CNOs) and metal-based nanoparticles, such as gold nanoparticles (AuNPs) and iron oxide (Fe₃O₄), were combined to generate a unique nanocomposite for the detection of CKMB. The immunosensor construction involved the deposition of the nanocomposite on the working electrode, followed by the incubation of an antibody and a blocking agent. Tris(2,2'-bipyridyl)-ruthenium(ii) chloride ([Ru(bpy)₃]²⁺Cl) was used as a luminophore, where tri-n-propylamine (TPrA) was selected as the co-reactant due to its aqueous immobility and luminescence properties. The analytical performance was demonstrated by cyclic voltammetry on ECL. The characterization of each absorbed layer was performed by cyclic voltammetry (CV) and chronocoulometry (CC) techniques in both EC and ECL. For further characterization of iron oxide, gold nanoparticles and carbon nano-onions, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were performed. The proposed immunosensor showcases a wide linear range (10 ng mL^-1 to 50 fg mL^-1), with an extremely low limit of detection (5 fg mL^-1). This CKMB immunosensor also exhibits remarkable selectivity, reproducibility, stability and resistance capability towards common interferences available in human serum. In addition, the immunosensor holds great potential to work with real serum samples for clinical diagnosis.

Fig. 1. Schematic for the fabrication of the label-free electrochemiluminescence CKMB immunosensor and the detection principle: (A) SWCNTs-SPE; (B) CNOs/Fe₃O₄/AuNPs/CS modified SPE; (C) anti-CKMB spiked immunosensor; (D) BSA blocked immunosensor; (E) immunosensor spiked with the target antigen (CKMB); (F) ECL signal.

Fig. 2. Nanocomposite characterization in EC and ECL using cyclic voltammetry and chronocoulometry: (A) ECL: CV line graph of (a) SWCNT-SPE, (b) CNO/SWCNT-SPE, (c) AuNP/CNO/SWCNT-SPE, (d) Fe₃O₄/AuNP/CNO/SWCNT-SPE, (e) CS/Fe₃O₄/AuNP/CNO/SWCNTs. (B) EC: CV line graph of (a) SWCNT-SPE, (b) CNO/SWCNT-SPE, (c) AuNP/CNO/SWCNT-SPE, (d) Fe₃O₄/AuNP/CNO/SWCNT-SPE, (e) CS/Fe₃O₄/AuNP/CNO/SWCNTs. (C) EC: CC bar diagram of (a) SWCNT-SPE, (b) CNO/SWCNT-SPE, (c) AuNP/CNO/SWCNT-SPE, (d) Fe₃O₄/AuNP/CNO/SWCNT-SPE, (e) CS/Fe₃O₄/AuNP/CNO/SWCNTs, (n = 3) at 21.5 ± 0.5 °C in 10 mM PBS, pH 7.4 (n = 3).

Fig. 3. SEM images of iron oxide (Fe₃O₄) (A), gold nanoparticles (AuNPs) (B) and carbon nano-onions (CNOs) (C) at 40 000×.

Fig. 4. TEM images of carbon nano-onions (CNOs at 5 nm) (A), gold nanoparticles (AuNPs at 5 nm) (B) and iron oxide (Fe₃O₄ at 20 nm) (C).

Fig. 5. Step-wise layer-by-layer characterization study of CKMB immunosensors using ECL (A) cyclic voltammetry: (a) SWCNT-SPE, (b) CNO/Fe₃O₄/AuNPs/CS/SWCNT-SPE, (c) Ab/CNO/Fe₃O₄/AuNP/CS/SWCNT-SPE, (d) Ag/BSA/Ab/CNO/Fe₃O₄/AuNP/CS/SWCNT-SPE measured in luminophore solution, CSE = 100 mV s⁻¹ using 1 : 100 [Ru(bpy)₃]Cl₂–TPrA solution containing 10 mM PBS, pH 7.4; and EC (B) chronocoulometry: (a) SWCNTs-SPE, (b) CNOs/Fe₃O₄/AuNP/CS/SWCNT-SPE, (c) Ab/CNO/Fe₃O₄/AuNP/CS/SWCNT-SPE, (d) Ag/BSA/Ab/CNOs/Fe₃O₄/AuNP/CS/SWCNT-SPE measured in redox probe (5 mM [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻) solution, CSE = 100 mV s⁻¹ at 21.5 ± 0.5 °C in 10 mM PBS, pH 7.4 (n = 3).

Fig. 6. Study of the analytical performance of the fabricated immunosensor in detecting CKMB: (A) CV line graph representing different concentrations of CKMB antigens from 10 ng mL⁻¹ to 50 fg mL⁻¹: (a) 10 ng mL⁻¹, (b) 1 ng mL⁻¹, (c) 500 pg mL⁻¹, (d) 100 pg mL⁻¹, (e) 10 pg mL⁻¹, (f) 1 pg mL⁻¹, (g) 0.5 pg mL⁻¹, (h) 0.1 pg mL⁻¹, (i) 0.05 pg mL⁻¹. (B) The calibration curves of the CKMB immunosensors towards different concentrations from 10 ng mL⁻¹ to 50 fg mL⁻¹ of CKMB, graph has been plotted as ECL intensity vs. log concentration for best fitting (n = 3).

Fig. 7. Analysis of reproducibility, stability, selectivity and interference-resistance of the immunosensor. (A) BSA/CNOs/Fe₃O₄/AuNPs/CS/SWCNTs-SPE immunosensors were incubated with 100 pg mL⁻¹ CKMB and their signals were recorded; (B) BSA/CNOs/Fe₃O₄/AuNPs/CS/SWCNTs-SPE immunosensor incubated with 100 pg mL⁻¹ CKMB and stored at 4 °C. Signals were recorded and assessed at five-day intervals to study the long-term stability of the immunosensors; (C) CKMB immunosensor was tested against other analytes known to be present in serum and the signal was recorded. The different samples tested were: (1) 100 pg mL⁻¹ CKMB; (2) 100 pg mL⁻¹ HP; (3) 100 pg mL⁻¹ HCG; (4) 100 pg mL⁻¹ leptin; (5) 100 pg mL⁻¹ CRP; (6) 100 pg mL⁻¹ CEA; (7) 100 pg mL⁻¹ cTnT; (8) 100 pg mL⁻¹ cTnI; (9) 100 pg mL⁻¹ cortisol; (10) 100 pg mL⁻¹ β-2M; (11) 100 pg mL⁻¹ AFP (n = 3); and (D) CKMB immunosensor was tested against 100 pg mL⁻¹ of CKMB in combination with 1000 pg mL⁻¹ of other analytes known to be present in serum and the signal recorded. The samples tested were (1) CKMB only, (2) CKMB + cTnT, (3) CKMB + cTnI, (4) CKMB + β-2M, (5) CKMB + cTnT + cTnI + β-2M (n = 3).