A Hemin-Graphene Nanocomposite-Based Aptasensor for Ultrasensitive Colorimetric Quantification of Leukaemia Cells Using Magnetic Enrichment

Abstract

Diagnostic blood cell counting is of limited use in monitoring a minimal number of leukaemia cells, warranting further research to develop more sensitive and reliable techniques to identify leukaemia cells in circulation. In this work, a hemin-graphene nanocomposite-based aptasensor was developed for ultrasensitive colorimetric detection of leukaemia cells (CEM) using magnetic enrichment. Hemin-conjugated graphene oxide nanocomposites (HGNs) were prepared by hydrazine reduction using graphene oxide nanosheets and hemins. Hence, the prepared HGNs become able to absorb single-stranded DNA and acquire peroxidase-like activity. The aptamer sgc8c, which recognizes a specific target on leukaemia cells, was absorbed onto HGNs to capture the target CEM cancer cells. The captured target cells that associated with the HGNs were then concentrated and separated by magnetic beads (MBs) coated with sgc8c aptamers, forming a HGN-cell-MB sandwich structure. These sandwich structures can be quantified via an oxidation reaction catalysed by HGNs. By utilizing dual signal amplification effects generated by magnetic enrichment and the improved peroxidase activity of HGNs, the biosensor allowed for highly sensitive detection of 10 to 10⁵ CEM cells with an ultra-low limit of detection (LOD) of 10 cells under optimal conditions. It is expected that the proposed aptasensor can be further employed in monitoring the minimal residual disease during the treatment of leukaemia.

Keywords: biosensors; graphene oxide; hemin; leukaemia cells; sgc8c aptamer.

Figure 1

Schematic illustration of the principle for the detection of leukaemia cells based on hemin–graphene nanocomposites (HGNs) and magnetic enrichment.

Figure 2

TEM and AFM analysis of GO and HGNs. TEM images of graphene oxide nanosheets (A) and hemin–graphene nanocomposites (HGNs) (B), scale bars are 500 nm long. AFM images of graphene oxide (C) and HGNs (D) show detailed surface topographies of the nanosheets before and after conjugation with hemin, respectively; cross-sectional analyses identified by the lines as graphs in AFM images shows the heights of GO (E) and HGNs (F).

Figure 3

Characterization and analyses of HGNs to assess the conjugation of hemin onto the reduced graphene oxide. UV (A) and IR (B) absorption spectra of HGNs are compared with those of hemin and graphene oxide. Surface scanning patterns of graphene oxide and HGNs by X-ray photoelectron spectroscopy are shown in (C) and (E), respectively, and their carbon spectra are shown in (D) and (F), respectively.

Figure 4

Optimization of the experimental conditions. (A) The signal-to-background ratios (S/N) of the aptasensor for detection of the same number of CEM cells with HGNs was prepared by varying the hemin-to-GO weight ratio from 1 to 10. (B) Optimization of the concentration of capture probes for preparing functionalized magnetic beads to determine CEM cells. Dependence of the concentration of TMB (C,D) H₂O₂ on the peroxidase-like activity of HGNs with the hemin-to-GO weight ratio of 5.

Figure 5

Application of the HGN-based aptasensor for quantification of CEM cells. (A) The UV-vis absorption spectra recorded by the HGN-based aptasensors for detecting various concentrations of target CEM cells with cell amount from 0 to 10⁵. (B) The relationship between absorbance intensity and the cell concentration. The inset shows the linear calibration curve between absorbance intensity and the logarithm of cell number. (C) Absorbance intensity ratio of control cells (including Jurkat, RAMOS, A549 and HepG2 cells) relative to the target CEM cells determined by the aptasensor.