Automated Support-Resolution Strategy for a One-Way Chemiluminescent Multiplex Immunoassay

Abstract

An automated support-resolution strategy was designed to couple with a flow-through immunosensing system for performing a one-way chemiluminescent (CL) multiplex immunoassay. Different from multilabel and multichannel-based detection techniques, this immunoassay method employed a single horseradish peroxidase (HRP) label in one way. With the use of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes, the capture antibodies for CEA and AFP were immobilized on the inner wall of a glass tube and the surface of paramagnetic microspheres (PMs), respectively. The on-line incubation could be performed in the glass tube after introducing the mixture of CEA, HRP-labeled anti-CEA antibody, AFP, anti-AFP immobilized PMs, and HRP-labeled anti-AFP antibody. With the use of a wash step, the formed sandwich immunocomplexes were separated automatically and the immunocomplex immobilized PMs were captured in another unmodified glass tube with a magnet. The CL signals from the two glass tubes were near-simultaneously collected with the aid of an optical shutter to perform quantitative detection. CEA and AFP could be rapidly assayed in the ranges of 1.0-60 and 1.0-80 ng/mL within 27 min. The assay results of clinical serum samples with the proposed method were in an acceptable agreement with the reference values. This system provides a promising multiplex immunoassay approach for clinical applications.

Electrochemiluminescence immunosensor for α-fetoprotein using Ru(bpy)32+-encapsulated liposome as labels

“…The regression equation was I ECL = 592 + 44629 C (pg/mL), where I was the ECL intensity, and c was the AFP concentration. An extremely low detection limit of 0.001 pg/mL was obtained, much lower than that in previous reports [10][11][12]. In addition, a liner range from 0.05 to 0.2 pg/mL (n = 4) and a detection limit of 0.01 pg/mL was obtained with the immunosensor fabricated without GNPs, suggesting that GNPs contribute to the high sensitivity of the immunosensor due to the large surface area for high loading of antibody [14].…”

“…1% (v/v) Triton X-100 solution was used to open the liposome and the released Ru(bpy) 3 2+ , which was proportional to the amount of AFP, was detected with ECL method in the presence of 2-di-n-butylaninoethanol (DBAE). Under optimized conditions, a linear range of AFP from 0.005 to 0.2 pg/mL and an extremely low detection limit of 0.001 pg/mL was obtained, much lower than that in previous reports [10][11][12]. The proposed ECL immnuosensor exhibited high sensitivity, specificity, and good stability, which may find promising applications in clinical analysis.…”

“…The average concentration of AFP in normal adults is less than 20 ng/mL and AFP levels in serum often increase under disease conditions [9]. Since detecting AFP levels in serum is important for clinical diagnosis, several different immunological methods for AFP detection have been proposed [10][11][12]. However, ultrasensitive methods for detecting low levels of AFP are still challenging.…”

Recent Developments in Emerging Microimmunoassays

“…While most static support immunoassay systems employ fluorescence detection, the Ju group investigated chemiluminescent immunoassay platforms for the near-simultaneous detection of two analytes, CEA and AFP [61,62]. In the first study, a modified glass tube with immobilized anti-CEA antibody was incubated with a mixture of the two antigens, their horseradish peroxidase-conjugated detection antibodies, and anti-AFP immobilized on paramagnetic particles [61].…”

Nanoparticle-Based Sandwich Electrochemical Immunoassay for Carbohydrate Antigen 125 with Signal Enhancement Using Enzyme-Coated Nanometer-Sized Enzyme-Doped Silica Beads

“…Phone/Fax: +86 591 2286 6135. hard magnetic material, were extensively applied in the nanoparticle-based assays since they have good biocompatibility and can be separated very readily from reaction mixtures with the help of an external magnetic field. [20][21][22][23] The functionalized probes could pull antibodies bound to magnetic nanoparticles from one laminar flow path to another by applying a local magnetic field gradient and selectively removing them from flowing biological fluids without any washing step. Moreover, it has been successfully applied as matrixes for the electrochemical detection of biomarkers in our past works.…”