A novel reverse fluorescent immunoassay approach for sensing human chorionic gonadotropin based on silver-gold nano-alloy and magnetic nanoparticles

Abstract

A novel and environmentally friendly reverse fluorescent immunoassay approach was proposed and utilized for sensing human chorionic gonadotropin (HCG) in human serum by coupling a newly prepared and highly fluorescent glutathione-stabilized silver-gold nano-alloy (GSH-AgAuNAs) with magnetic nanoparticles (MNPs). To construct such a reverse system, fluorescent GSH-AgAuNAs and MNPs were first prepared and bio-functionalized with monoclonal antibodies (Mab-I and Mab-II) toward HCG antigen, respectively. Then, the GSH-AgAuNAs functionalized with Mab-I were incubated with HCG, followed by the addition of MNPs attached to Mab-II. Thereafter, a sandwich-type immunoassay could be constructed for determination of HCG owing to the antibody-antigen recognition between the functionalized GSH-AgAuNAs and MNPs. Afterwards, a magnetic collection was employed. Hence, the amount of GSH-AgAuNAs would be reduced through an immuno-magnetic separation, thus weakening the fluorescent intensity. Different from conventional immunoassay, our work determined the quantitative signal by measuring the decreasing gradient fluorescent intensity. Under optimal conditions, the developed reverse method exhibited a wide linear range of 0.5-600 ng mL(-1) toward HCG with a detection limit of 0.25 ng mL(-1). Additionally, the proposed immunoassay was validated using spiked samples, illustrating a satisfactory result in practical application.

Chemically Regulated ROS Generation from Gold Nanoparticles for Enzyme-Free Electrochemiluminescent Immunosensing

“…MBs were modified with anti-IgA antibodies according to a protocol from previous research . In brief, 200 nm MBs activated with N -hydroxysuccinimide (NHS) were first washed with methanol by centrifugation for 5 min at 15000 rpm and 4 °C.…”

Electrochemiluminscence Based Biosensors with AuNP Showing Catalytic ROS Generation

“…MBs were modified with anti-IgA antibodies according to a protocol from previous research [6]. In brief, MBs activated with NHS (200 nm, Tamagawa Seiki) were washed with methanol by centrifugation for 5 min at 15,000 rpm and 4 °C.…”

“…AuNPs were also modified with anti-IgA antibodies according to a protocol from previous research [6]. 100 μL anti-IgA was added to 10 mL of AuNPs dispersed in PBS under stirring for 24 h at 4 °C.…”

“…AuNPs are widely known to catalyze various reactions such as the oxidation of carbon monoxide to carbon dioxide. [6] Our purposes are to use AuNP catalysis for the high efficiency generation of ROS, and to measure the generated ROS by ECL. To this end, we investigated the mechanism of AuNP catalyzed ROS generation.…”

Magnetic-bead-based sub-femtomolar immunoassay using resonant Raman scattering signals of ZnS nanoparticles

“…They can be manipulated remotely via external magnetic field, thus facilitating greatly the concentration, separation, and purification processes in immunoassays. They have been incorporated into multiple immunoassay strategies to improve the detection performance [6][7][8][9]. For example, Duan et al utilized magnetic beads as a solid support for antibody in a novel laser-induced fluorescence immunoassay, and the detection limit obtained for α-fetoprotein was as low as 1.6 pg/ml, which is about three orders of magnitude lower than that of conventional ELISA [9].…”