Signal Amplification Electrochemical Immunosensor Based On Envision Copolymers

This paper built a series of signal tags for signal amplification based on EnVision copolymers.The study of the thesis mainly from the preparation of nano-metals labeled signal tags, theconstruction of immunesensor and signal amplification. The details performed as the follows:1. An ultrasensitive electrochemical immunosensor for alpha-fetoprotein using an Envisioncomplex-antibody copolymer as a sensitive label.A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP), using ahorseradish peroxidase (HRP)-functionalized EnVision antibody complex (EV, a large number ofsecondary antibodies and horseradish peroxidase (HRP) coupled to a dextran backbone) as thelabel. The sensor was constructed using the following steps: First, gold electrode (GE) wasmodified with nano-gold (AuNPs) by electro-deposition in HAuCl4solution. The high affinity ofthe AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next,the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1); these antibodiesattach to the thiolated protein G layer through their non-antigenic regions, leaving the antigenbinding sites for binding of target analyte. Following a sandwich immunoreaction, an EV-AFPAb2-AFP-AFP Ab1immunocomplex was formed on the electrode surface, allowing large amountsof HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ) inthe presence of hydrogen peroxide (H2O2). Highly amplified detection was achieved, with adetection limit of2pg mL-1and a linear range of5to200pg mL-1for AFP solutions. Thecorrelation coefficient was0.998for both linear ranges. Measurements of AFP in the serum ofcancer patients correlated strongly with standard enzyme-linked immunosorbent assays (ELISA).These easily fabricated EV-modified immunosensors show excellent promise for future fabricationof bioelectronic arrays.2. Incubation-free electrochemical immunoassay for diethylstilbestrol in milk using goldnanoparticle-antibody conjugates for signal amplification.In this paper, we reported on a novel and sensitive sandwich-type electrochemicalimmunosensor which was fabricated on a glassy carbon electrode (GCE) for ultra trace levels ofdiethylstilbestrol (DES) based on a novel enhanced sensing label, which was prepared by anorientation-controlled immobilization of the EV on the gold nanoparticles. The large amounts ofHRP on the label can catalyze and amplify the current signals at room temperature. Under the optimal conditions, the sensor can detect the concentration of DES from5to500pg mL-1with alow detection limit of2pg mL-1. The correlation coefficients was0.999for the linear range. Thedeveloped ultrasensitive electrochemical immunosensor can operate without incubation, which cangreatly shorten the detection time to5min. The proposed method has been successfully applied tothe detection of DES in milk samples with satisfactory results.3. Sensitive and incubation-free electrochemical immunosensor for DES detection in milkbased on triple signal amplification strategy of β-cyclodextrin and platinum nanoparticles labeledwith EV.A novel electrochemical immunosensor for sensitive detection of DES was described thatused a β-cyclodextrin (β-CD) sensor platform and the signal tag which was obtained byorientation-controlled immobilization of EV on platinum nanoparticles (PtNPs). Greatly enhancedsensitivity for DES was based on a triple signal amplification strategy: firstly, the synthesizedPtNPs, which yielded a narrow size and homogeneous distribution, allowed several binding eventsof EV on each PtNPs. Enhanced sensitivity was achieved by introducing the bioconjugates ofPtNPs labeled with EV onto the electrode surface through “sandwich-type” immunoreactions.When H2O2was added, PtNPs and HRP on EV copolymers can co-catalyze the reduction of H2O2,which can dual-amplify its reductive current. Secondly, β-CD was used to capture and enrich DESinstead of the primary antibodies for the biosensor platform, which can further amplify thedetection response. Thus triple signal amplification strategy was developed. Under optimizedconditions, the developed electrochemical immunosensor displayed good linearity from1to100ng mL-1toward DES standards with a low detection limit of0.3pg mL1(S/N=3). Thisimmunoassay was evaluated with milk samples, receiving good accordance with results fromELISA. In addition, this strategy has provided a promising potential method for DES detection inmilk samples.