Dual-mode fluorescent and colorimetric immunoassay for the ultrasensitive detection of alpha-fetoprotein in serum samples

Abstract We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 10 6 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 10 3 -fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3′,5,5′-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%–100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.