Ultrasensitive Multiplexed Immunoassay with Electrochemical Stripping Analysis of Silver Nanoparticles Catalytically Deposited by Gold Nanoparticles and Enzymatic Reaction

Abstract

A novel ultrasensitive multiplexed immunoassay method was developed by combining alkaline phosphatase (ALP)-labeled antibody functionalized gold nanoparticles (ALP-Ab/Au NPs) and enzyme-Au NP catalyzed deposition of silver nanoparticles at a disposable immunosensor array. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. After sandwich-type immunoreactions, the ALP-Ab/Au NPs were captured on an immunosensor surface to catalyze the hydrolysis of 3-indoxyl phosphate, which produced an indoxyl intermediate to reduce Ag(+). The silver deposition process was catalyzed by both ALP and Au NPs, which amplified the detection signal. The deposited silver was then measured by anodic stripping analysis in KCl solution. Using human and mouse IgG as model analytes, this multiplexed immunoassay method showed wide linear ranges over 4 orders of magnitude with the detection limits down to 4.8 and 6.1 pg/mL, respectively. Acceptable assay results for practical samples could be obtained. The newly designed strategy avoided cross talk and the need of deoxygenation for the electrochemical immunoassay and, thus, provided a promising potential in clinical applications.

Signal Amplification for Highly Sensitive Immunosensing

“…Through sandwich-type immunoreaction and biotin-streptavidin affinity reaction amplifying sensing signals, the proposed immunosensor showed wide linear ranges with the detection limits down to 0.024 and 0.032 pg mL -1 , respectively. Similarly, Ju's group [30] reported an ultrasensitive multiplexed immunosensing method developed by combination with an ALP-labeled antibody functionalized Au NP probe and the enzyme-Au NP catalyzed deposition of Ag NPs at a disposable immunosensor array (Fig. 7).…”

Surface Treatment Strategies on Catalytic Metal Nanoparticles

“…This strategy allows the generation of quantitative electrochemical signals which are correlated with the amount of target molecules captured via the biorecognition events that take place at the interface of the electrode. Particularly, enzyme-functionalized metal nanoparticles are of great interest because they can be also used as labels to improve the detection sensitivity by measuring the enzymatic catalysis of electroactive substrates on the surface of the electrode [119]. Also, interesting examples of the roles of metal nanoparticles modified with antibodies dealing with both the recognition of immunoreagents and the signal transduction/amplification processes to the detection of tumor markers and disease biomarkers in biological fluids such as urine, blood, or tissues should be included in this section [120,121].…”

Sensitive biosensing strategy based on functional nanomaterials

“…Such protocols rely on capturing the gold or silver nanoparticles to the hybridized target and use anodic-stripping voltammetry for electrochemical measuring the metal tracers. For example, an electrochemical method is employed for the Au-nanoparticle-based quantitative detection of the 406-base human cytomegalovirus DNA sequence (HCMV DNA) [30]; and a novel ultrasensitive multiplexed immunoassay method has been developed by combining alkaline phosphatase-labeled antibody functionalized gold nanoparticles and enzyme-Au NP catalyzed deposition of silver nanoparticles at disposable immunosensor array ( Figure 3) [31]. The deposited silver measured by anodic stripping analysis in KCl solution shows the amplified signal for multiplexed immunoassay in wide linear ranges over four orders of magnitude with the detection limits down to 4.8 pg/mL.…”