Ferritin-Triggered Redox Cycling for Highly Sensitive Electrochemical Immunosensing of Protein

Electrochemical immunoassay amplified with redox cycling has become a challenging topic in highly sensitive analysis of biomarkers. Here a ferritin-triggered redox cycling is reported by using a highly outersphere reaction-philic (OSR-philic) redox mediator ruthenium hexamine (Ru­(NH3)63+) to perform the OSR-philic/innersphere reaction-philic (ISR-philic) controlled signal amplification. The screened mediator can meet the needs of lower E0′ than ferritin, low reactivity with ISR-philic species, and quick electron exchange with ferritin redox couple. The ferritin-labeled antibody is first bonded to immunosensor surface by recognizing the target antigen capured by the immobilized primary antibody. The ferritin then mediates OSR-philic/ISR-philic transfer from Ru­(NH3)63+/2+/immunosensor to ferritin-H2O2 redox system. The fast mediation and excellent resistant of highly OSR-philic Ru­(NH3)63+ against radical oxygen species lead to highly sensitive electrochemical readout and high signal-to-background ratio. The proposed redox cycling greatly enhances the readout signal and the sensitivity of traditional ferritin-labeled sandwich immunoassay. Using Enteropathogenic coli (E. coli) antigen as a model analyte, the developed method shows excellent linearity over the concentration range from 10.0 pg/mL to 0.1 μg/mL and a detection limit of 10.0 fg/mL. The acceptable accuracy, good reproducibility, and selectivity of the proposed immunoassay method in real samples indicate the superior practicability of the ferritin-triggered redox cycling

A Tyrosinase-Responsive Nonenzymatic Redox Cycling for Amplified Electrochemical Immunosensing of Protein

Electrochemical immunoassays with high sensitivity and a wide dynamic range are still a challenge in clinical diagnosis. This study reports a novel tyrosinase (Tyr)-responsive nonenzymatic redox cycling for significantly amplifying the electrochemical signal produced from Tyr-labeled immunoassays. The tyrosinase could be captured on the immunosensor surface by a sandwich immunoreaction. The immunosensor was conveniently prepared by covalently binding capture antibody to a chitosan-coated electrode. Using phenol as a substrate and NADH as reducing agent, which showed negligible background due to low electroactivity of phenol and high oxidation overpotential of NADH, the oxygenation activity of tyrosinase could convert poorly electroactive phenol to highly electroactive catechol to trigger an NADH-related nonenzymatic electrochemical-chemical (EC) catalysis. The EC redox cycling leads to a high signal-to-noise ratio for immunoassays. Using carcinoembryonic antigen (CEA) as an analyte model, the amplified immunoassay showed excellent performance with a detectable range from 1.0 pg/mL to 0.1 μg/mL and a sub-pg/mL-level detection limit. The acceptable accuracy and good reproducibility of the proposed immunoassay method indicated its superior suitability in clinical diagnosis

“Outer-Sphere to Inner-Sphere” Redox Cycling for Ultrasensitive Immunosensors

This paper reports chemical–chemical (CC) and electrochemical–chemical–chemical (ECC) redox cycling, for use in ultrasensitive biosensor applications. A triple chemical amplification approach using an enzymatic reaction, CC redox cycling, and ECC redox cycling is applied toward electrochemical immunosensors of cardiac troponin I. An enzymatic reaction, in which alkaline phosphatase converts 4-aminophenyl phosphate to 4-aminophenol (AP), triggers CC redox cycling in the presence of an oxidant and a reductant, and electrochemical signals are measured with ECC redox cycling after an incubation period of time in an air-saturated solution. To obtain high, selective, and reproducible redox cycling without using redox enzymes, two redox reactions [the reaction between AP and the oxidant and the reaction between the oxidized form of AP (4-quinone imine, QI) and the reductant] should be fast, but an unwanted reaction between the oxidant and reductant should be very slow. Because species that undergo outer-sphere reactions (OSR-philic species) react slowly with species that undergo inner-sphere reactions (ISR-philic species), highly OSR-philic Ru­(NH₃)₆³⁺ and highly ISR-philic tris­(2-carboxyethyl)­phosphine (TCEP) are chosen as the oxidant and reductant, respectively. The OSR- and ISR-philic QI/AP couple allows fast redox reactions with both the OSR-philic Ru­(NH₃)₆³⁺ and the ISR-philic TCEP. Highly OSR-philic indium–tin oxide (ITO) electrodes minimize unwanted electrochemical reactions with highly ISR-philic species. Although the formal potential of the Ru­(NH₃)₆³⁺/Ru­(NH₃)₆²⁺ couple is lower than that of the QI/AP couple, the endergonic reaction between Ru­(NH₃)₆³⁺ and AP is driven by the highly exergonic reaction between TCEP and QI (via a coupled reaction mechanism). Overall, the “outer-sphere to inner-sphere” redox cycling in the order of highly OSR-philic ITO, highly OSR-philic Ru­(NH₃)₆³⁺/Ru­(NH₃)₆²⁺ couple, OSR- and ISR-philic QI/AP couple, and highly ISR-philic TCEP allows high, selective, and reproducible signal amplification. The electrochemical data obtained by chronocoulometry permit a lower detection limits than those obtained by cyclic voltammetry. The detection limit of an immunosensor for troponin I in serum, calculated from the anodic charges in chronocoulometry, is ca. 10 fg/mL

Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical–Chemical–Chemical Redox Cycling for the Detection of <i>E. coli</i> O157:H7

Signal amplification by enzyme labels in enzyme-linked immunosorbent assays (ELISAs) is not sufficient for detecting a low number of bacterial pathogens. It is useful to employ approaches that involve multiple signal amplification such as enzymatic amplification plus redox cycling. An advantageous combination of an enzyme product [for fast electrochemical–chemical–chemical (ECC) redox cycling that involves the product] and an enzyme substrate (for slow side reactions and ECC redox cycling that involve the substrate) has been developed to obtain a low detection limit for E. coli O157:H7 in an electrochemical ELISA that employs redox cycling. In our search for an alkaline phosphatase substrate/product couple that is better than the most common couple of 4-aminophenyl phosphate (APP)/4-aminophenol (AP), we compared five couples: APP/AP, hydroquinone diphosphate (HQDP)/hydroquinone (HQ), l-ascorbic acid 2-phosphate/l-ascorbic acid, 4-amino-1-naphthyl phosphate/4-amino-1-naphthol, and 1-naphthyl phosphate/1-naphthol. In particular, we examined signal-to-background ratios in ECC redox cycling using Ru­(NH3)63+ and tris­(2-carboxyethyl)­phosphine as an oxidant and a reductant, respectively. The ECC redox cycling that involves HQ is faster than the cycling that involves AP, whereas the side reactions and ECC redox cycling that involve HQDP are negligible compared to the APP case. These results seem to be due to the fact that the formal potential of HQ is lower than that of AP and that the formal potential of HQDP is higher than that of APP. Enzymatic amplification plus ECC redox cycling based on a HQDP/HQ couple allows us to detect E. coli O157:H7 in a wide range of concentrations from 103 to 108 colony-forming units/mL

Optimization of Phosphatase- and Redox Cycling-Based Immunosensors and Its Application to Ultrasensitive Detection of Troponin I

The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium–tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl2, and the optimum conditions for antigen–antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen–antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (l-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (l-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors