A renewable and ultrasensitive electrochemiluminescence immunosenor based on magnetic RuL@SiO2-Au~RuL-Ab2 sandwich-type nano-immunocomplexes

Abstract

An ultrasensitive and renewable electrochemiluminescence (ECL) immunosensor was developed for the detection of tumor markers by combining a newly designed trace tag and streptavidin-coated magnetic particles (SCMPs). The trace tag (RuL@SiO(2)-Au~RuL-Ab2) was prepared by loading Ru(bpy)(3)(2+)(RuL)-conjuged secondary antibodies (RuL-Ab2) on RuL@SiO(2) (RuL-doped SiO(2)) doped Au (RuL@SiO(2)-Au). To fabricate the immunosensor, SCMPs were mixed with biotinylated AFP primary antibody (Biotin-Ab1), AFP, and RuL@SiO2-Au~RuL-Ab2 complexes, then the resulting SCMP/Biotin-Ab1/AFP/RuL@SiO2-Au~RuL-Ab2 (SBAR) sandwich-type immunocomplexes were absorbed on screen printed carbon electrode (SPCE) for detection. The immunocomplexes can be easily washed away from the surface of the SPCE when the magnetic field was removed, which made the immunosensor reusable. The present immunosensor showed a wide linear range of 0.05-100 ng mL(-1) for detecting AFP, with a low detection limit of 0.02 ng mL(-1) (defined as S/N = 3). The method takes advantage of three properties of the immunosensor: firstly, the RuL@SiO(2)-Au~RuL-Ab2 composite exhibited dual amplification since SiO(2) could load large amount of reporter molecules (RuL) for signal amplification. Gold particles could provide a large active surface to load more reporter molecules (RuL-Ab2). Accordingly, through the ECL response of RuL and tripropylamine (TPA), a strong ECL signal was obtained and an amplification analysis of protein interaction was achieved. Secondly, the sensor is renewable because the sandwich-type immunocomplexes can be readily absorbed or removed on the SPCE's surface in a magnetic field. Thirdly, the SCMP modified probes can perform the rapid separation and purification of signal antibodies in a magnetic field. Thus, the present immunosensor can simultaneously realize separation, enrichment and determination. It showed potential application for the detection of AFP in human sera.

Keywords: [Ru(bpy)3]2+@SiO2-Au; alpha-fetoprotein; electrochemiluminescence; sandwich-type immunoreaction; screen printed carbon electrode.

Figure 1.

TEM images of (A) RuL@SiO₂; (B) RuL@SiO₂-Au.

Figure 2.

Effect of the different concentrations of TPA on the ECL intensity. Experimental parameters: initial potential = 0.0 V, high potential = 1.2 V, scan rate = 100 mV/s.

Figure 3.

ECL-potential curves of, (a) SCMP/Biotin-Ab1/AFP/RuL@SiO₂-Au∼RuL-Ab2; (b) SCMP/Biotin-Ab1/AFP/RuL@SiO₂-Au∼Ab2; and (c) SCMP/Biotin-Ab1/AFP/RuL-Ab2 in pH 7.4 PBS containing 10⁻⁵ M TPA. Experimental parameters: initial potential = 0.0 V, high potential = 1.2 V, scan rate = 100 mV/s.

Figure 4.

The schematic illustration of the ECL intensity versus the concentration of AFP (0.05 to 100 ng mL⁻¹) in 0.1 M PBS (pH 7.4) containing 10⁻⁵ M TPA. Insert: the relationship between Log of ΔECL signal towards log of different AFP concentrations. Experimental parameters: initial potential = 0.0 V, high potential=1.2 V, scan rate = 100 mV/s.

Figure 5.

Selectivity analysis of the ECL immunosensor in the presence of different interferents. The concentrations of the interferents were: CEA (10 ng mL⁻¹), HIgG (1 μg mL⁻¹), CA19-9(10 ng mL⁻¹), HCG (10 ng mL⁻¹), BSA (1 μg mL⁻¹), DA (1 μg mL⁻¹), L-lysine (1 μg mL⁻¹).

Figure 6.

Cyclic voltammograms performed in 1 mM Fe(CN)₆³⁻ solution (0.1 M KCl) at 100 mV s⁻¹ for (a) bare; (b) SBAR sandwich-type immunocomplexes; (c) removing the magnet and rinsing with PBST.

Scheme 1.

Schematic illustration of the preparing procedures of RuL@SiO₂ nanoparticles and RuL@SiO₂-Au composite.

Scheme 2.

Schematic illustration of the preparing procedures of RuL@SiO₂-Au∼RuL-Ab2 and RuL@SiO₂-Au∼Ab2.

Scheme 3.

Schematic illustration of the preparing procedures of SBAR sandwich-type immunocomplexes (design and process).

Scheme 4.

(a) Schematic illustration of three-electrode SPCE system; (b) The process of ECL measurements; (c) the ECL measurement system.