Gold nanoparticle enhanced hybridization chain reaction as a method for signal amplification. Application to electrochemical immunodetection of the ovarian cancer biomarker carbohydrate antigen 125.

Abstract

The authors describe a method for enhancing the hybridization chain reaction (HCR) by using gold nanoparticles (AuNPs). This can considerably improve the sensitivity of electrochemical immunoassays as demonstrated for the carbohydrate antigen 125 (CA125), a biomarker for ovarian cancer. Compared to previous HCR based assays, the DNA acting as fuel strands were immobilized onto AuNPs, so that dendrimeric like chains were formed on the electrode after HCR. The improved signal is due to the reaction of DNA on the electrode. Specifically, the reaction of the phosphate groups of DNA with molybdate forms redox-active molybdophosphate, and this generates a strong electrochemical current. The immunosensor was prepared by sequential capturing, on the electrode, (a) antibody against CA125, (b) analyte (CA125), and (c) an aptamer against CA125 to form a sandwich structure. The primer on the aptamer sequence initiates HCR by annealing to one strand of DNA on the AuNPs and to another DNA in solution. The increased loading of DNA molecules onto the electrode increases the amount of phosphate groups and subsequently increases the electrical signal. The sensitivity of the assay is found to be significantly improved compared to assays without HCR and when using conventional HCR. The immunosensor was successfully applied to the determination of CA125 in human serum samples. The detection limit (based on an S/N ratio of 3) is 50 μU.mL-1. This indicates that this signal amplification strategy has a large potential in terms of clinical applications. It may be modified such that it also can be applied to the determination of other analytes for which proper aptamers are available. Graphical abstract Gold nanoparticle (AuNP) enhanced hybridization chain reaction is reported to improve the sensitivity of electrochemical immunosensor. Hybridization chain reaction is carried out by annealing of H1 DNA strand immobilized on AuNP to the sticky end primer sequence of the aptamer and H2 strand to the complementary sequence of H1.