Flexible electrical aptasensor using dielectrophoretic assembly of graphene oxide and its subsequent reduction for cardiac biomarker detection

Abstract

Cardiac troponin T (cTnT) is considered a clinical standard for its high specificity and sensitivity when diagnosing acute myocardial infarction; however, most studies on the electrical sensors of cardiac troponin biomarkers have focused on cTnI rather than cTnT. This study presents label-free, low-cost, transparent, and flexible aptamer-based immunosensors for the electrical detection of cTnT using reduced graphene oxide (rGO) sheets. GO was first deposited by AC dielectrophoresis between two predefined source and drain electrodes on a 3-aminopropyltriethoxylsilane-modified polyethylene terephthalate substrate. The GO was then reduced using hydrazine vapour without damaging the substrate, resulting in uniform, controlled, and stable deposition of rGO sheets, and demonstrating more stability than those directly deposited by dielectrophoresis. Amine-modified single-strand DNA aptamers against cTnT were immobilized onto the rGO channels. The relative resistance change of this sensor owing to the attachment of cTnT was quantified as the cTnT concentration decreased from 10 ng/mL to 1 pg/mL in phosphate buffered saline (PBS) and 10-fold diluted human serum in PBS, with the limits of detection being 1.2 pg/mL and 1.7 pg/mL, respectively, which is sufficiently sensitive for clinical applications. High-yield and rapid fabrication of the present rGO sensors will have significant influences on scaled-up fabrication of graphene-based sensors.

Figure 1

(A) A schematic of the DEP-deposition of GO sheets on an APTES-modified PET substrate. (B) Schematic of the rGO aptasensors for the detection of cTnT, which consisted of rGO sheets (DEP-deposition of GO and its subsequent reduction) and cTnT aptamers on an APTES-modified PET substrate. (C) Schematic of cTnT aptamer immobilization on the graphene surfaces via PBSE linker and cTnT aptamers. Field emission scanning electron microscopy (FE-SEM) images of DEP-assembled thin layers of GO sheets (D) before and (E) after reduction via hydrazine vapour between two Cr/Au electrodes, (F) direct DEP deposition of rGO sheets, and (G) the immobilized cTnT aptamers on the rGO surface. (H) Fluorescence image of the immobilized cTnT aptamers on the rGO surface.

Figure 2

AFM images of DEP-deposited GO (A) and its reduced sheets (rGO) (B) between the two Cr/Au electrodes. (C) AFM image of the rGO surface. Lines 1, 2, and 3 represent the measuring lines between the Cr/Au electrodes. (D) Height profiles of the rGO sheets along the three lines in (C) measured by AFM.

Figure 3

Relative resistance change (RRC) of the aptasensors as the concentration of cTnT in PBS (pH 7.4, 1×) and 10-fold-diluted human serum was varied from 1 pg mL⁻¹ to 10 ng mL⁻¹. The error bars indicate the standard deviations of the measurements. The inset shows the measurements from 1 pg mL⁻¹ to 1 ng mL⁻¹.

Figure 4

Selectivity test of the rGO aptasensors using PBS (pH 7.4, 1×), 10-fold-diluted human serum in PBS (pH 7.4, 1×), cTnI (1 µg mL⁻¹), Myoglobin (1 µg mL⁻¹) and cTnT (10 pg mL⁻¹) in the two media. The error bars indicate the standard deviations of the measurements. One-way analysis of variance (ANOVA) was used to check whether the measured RRCs were significantly different. (ns: p > 0.05 and ***p < 0.0001).

Figure 5

Reusability of the aptasensor. Relative resistance change (RRC) of the aptasensors at two concentrations of cTnT (10 pg mL⁻¹ to 100 pg mL⁻¹) in PBS (pH 7.4, 1×). The error bars indicate the standard deviations of the measurements.