Binary MoS2 nanostructures as nanocarriers for amplification in multiplexed electrochemical immunosensing: simultaneous determination of B cell activation factor and proliferation-induced signal immunity-related cytokines

Abstract

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS₂/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H₂O₂ system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL^-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies.

Keywords: APRIL; Amperometry; BAFF; CRC; Dual immunoplatform; MoS2/MWCNTs; SLE; Serum.

Fig. 1

Schematic display of the different steps involved in the preparation of the MWCNTs/MoS₂(-HRP)-dAb nanocarriers (a) and of the dual immunosensor constructed for the determination of BAFF and APRIL biomarkers using amperometric transduction (b)

Fig. 2

SEM images of (a) MoS₂ (inset at higher magnification), (b) o-MWCNTs, (c) GO (inset at higher magnification), (d) MoS₂/MWCNTs, (e) MoS₂/rGO/MWCNTs, and (f) MoS₂/GO

Fig. 3

TEM images of: a MoS₂, b MoS₂/MWCNTs (inset: o-MWCNTs), c MoS₂/GO (inset: GO), and d MoS₂/rGO/MWCNTs

Fig. 4

(a) Raman spectra and (b) zoom at high frequencies of MWCNTs (black), MoS₂/MWCNTs (red), and MoS₂/rGO/MWCNTs (blue) nanocomposites at λ_exc = 532 nm

Fig. 5

UV spectra of (1) GO, (2) o-MWCNTs, (3) MoS₂, (4) MoS₂/rGO, (5) MoS₂/MWCNTs, and (6) MoS₂/rGO/MWCNTs

Fig. 6

Photographs showing the peroxidase activity of MoS₂ nanocomposites in 250 μL of the TMB/H₂O₂ ready to use commercial solution

Fig. 7

Cyclic voltammograms (a) and Nyquist plots (b) recorded for 5 mM [Fe(CN)₆]^−3/−4 in 0.1 M KCl solutions for (1) bare SPCE, (2) MoS₂/SPCE, (3) MoS₂/GO/SPCE, (4) MoS₂/MWCNTs/SPCE, and (5) MoS₂/GO/MWCNTs/SPCE. a ν = 50 mV s⁻¹ and b range of frequencies: 10⁵–0.04 Hz; open circuit. The equivalent circuits used to adjust the experimental results are shown below

Fig. 8

Calibration plots constructed with the dual immunosensor for BAFF (white points) and APRIL (black points) using MoS₂/MWCNTs(-HRP)-dAb as carrier tags for the amperometric measurements at − 0.20 V vs. Ag pseudo-reference electrode. Other working conditions are summarized in Table 1

Fig. 9

Amperometric responses measured with the dual immunosensor for 0- (white bar) and 10-ng mL⁻¹ BAFF or 4-ng mL⁻¹ APRIL (grey bar) standards prepared in absence and in the presence of 1 mg mL⁻¹ hIgG, 50 mg mL⁻¹ HSA, 5 mg mL⁻¹ BSA, 5 mg mL⁻¹ HB, 30 pg mL⁻¹ NfL, 5 pg mL⁻¹ TAU, 1 ng mL⁻¹ TDP-43, 500 ng mL⁻¹ CDH-17, 10 ng mL⁻¹ IL13Rα2, 100 pg mL⁻¹ TNFα, 4 ng mL⁻¹ APRIL, or 1 ng mL⁻¹ BAFF

Fig. 10

BAFF (a) and APRIL (b) concentrations determined with the dual immunosensors for serum samples grouped into pools of healthy individuals and SLE and CRC patients. Real amperograms obtained for representative serum samples of a healthy individual and patients diagnosed with SLE and CRC are displayed

Fig. 11

Correlation plots between the BAFF (a) and APRIL (b) concentrations determined with the developed dual inmunosensors and the ELISA methodologies for both the analyzed cellular lysates and serum samples. The three replicates made for each sample are included in the plots