Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly

Abstract

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with Nmethyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag + from the C−Ag + −C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag + , respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color-and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

Programmable Nanostructures Based on Framework-DNA for Applications in Biosensing

“…However, the simultaneous detection of two biomarkers remains challenging. By using an aptamer to capture the target protein, it is possible to detect both alpha-fetoprotein (AFP) and glypican-3 (GPC3) in clinical specimens of hepatocellular carcinoma [ 101 ], also illustrated in Figure 4 a. This design used N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters and simple enzyme-free parallel CHA as an amplification strategy.…”

“… ( a ) Dual CHA system, with the released individual aptamer for the simultaneous analysis of AFP and GPC3 [ 101 ]. (Reprinted from ACS Applied Materials and Interfaces , Chen, P.; Jiang, P.; Lin, Q.; Zeng, X.; Liu, T.; Li, M.; Yuan, Y.; Song, S.; Zhang, J.; Huang, J.; et al Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly, 2022 , 14 , 28697–28705, Copyright (2022), with permission from the American Chemical Society).…”

A label-free electrochemical aptasensor based on platinum@palladium nanoparticles decorated with hemin-reduced graphene oxide as a signal amplifier for glypican-3 determination

“…(3) The multifunctional H-rGO-Pt@Pd NPs with a large specific surface area and excellent conductivity were not only used as an effective supporting platform for GPC3 aptamer immobilization but also as a sensitive electrical signal probe. (4) The specific binding between the aptamer and its target realizes high sensitivity and low detection limit of GPC3 detection.…”

“…The increased GPC3 expression in HCC indicates the sensitivity and specificity of HCC diagnosis. 3,4 Moreover, some patients with negative alpha-fetoprotein (AFP) serum tests were positive in GPC3 serum tests with high sensitivity. 5 Therefore, GPC3 has been identified as the most promising biomarker of HCC diagnosis and immunotherapy.…”

A Paper-Based Photoelectrochemical Sensing Platform Based on In Situ Grown ZnO/ZnIn2S4 Heterojunctions onto Paper Fibers for Sensitively Detecting AFP

“…With these considerations, it is highly reasonable to develop a paper-based immunoassay strategy for the accurate quantification of AFP targets, in order to meet the pressing need of practical diseases diagnosis, especially in resource-limited regions, where the medical infrastructures are limited. To date, approaches such as electrochemiluminescence, electrochemical, and fluorescence have been proposed for the timely detection of AFP [ 32 , 33 , 34 , 35 ]. There is no doubt that those approaches have made significant progress in the fields of clinical applications.…”