Investigation of dual-layer membrane cloaking method by surface plasmon resonance for direct chronoamperometric immunoassay of serum sample
A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.
- [Show abstract] [Hide abstract] ABSTRACT: The long-term stability of sensing interfaces is an important issue in biosensor fabrication. A novel stable gold nanoparticle (AuNP)-modified glassy carbon (GC) electrode interface (GC-Ph-AuNP)-based biosensor for detecting carcinoembryonic antigen (CEA) was developed. GC electrodes were modified with 1,4-phenylenediamine to form a stable layer, and then AuNPs were bound onto the GC electrodes through CAu bonds. Anti-CEA was directly adsorbed on AuNPs fixed on the GC electrode. The linear range of the immunosensor was from 10 fg to 100 ng mL(-1) with a detection limit of 3 fg mL(-1) (S/N=3). The current of the immunosensor was increased by 4% after one month. The GC-Ph-AuNP immunosensor showed high sensitivity, a wide linear range, low detection limit, and good selectivity and stability. The immobilization method of the immunosensor could be widely applied to construct other immunosensors.0Comments 28Citations