Kyungmin Jo

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 MgCl(2), 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.

Nanocatalyst-Based Assay Using DNA-Conjugated Au Nanoparticles for Electrochemical DNA Detection

Compared to enzymes, Au nanocatalysts show better long-term stability and are more easily prepared. Au nanoparticles (AuNPs) are used as catalytic labels to achieve ultrasensitive DNA detection via fast catalytic reactions. In addition, magnetic beads (MBs) are employed to permit low nonspecific binding of DNA-conjugated AuNPs and to minimize the electrocatalytic current of AuNPs as well as to take advantage of easy magnetic separation. In a sandwich-type electrochemical sensor, capture-probe-conjugated MBs and an indium-tin oxide electrode modified with a partially ferrocene-modified dendrimer act as the target-binding surface and the signal-generating surface, respectively. A thiolated detection-probe-conjugated AuNP exhibits a high level of unblocked active sites and permits the easy access of p-nitrophenol and NaBH 4 to these sites. Electroactive p-aminophenol is generated at these sites and is then electrooxidized to p-quinoneimine at the electrode. The p-aminophenol redox cycling by NaBH 4 offers large signal amplification. The nonspecific binding of detection-probe-conjugated AuNPs is lowered by washing DNA-linked MB-AuNP assemblies with a formamide-containing solution, and the electrocatalytic oxidation of NaBH 4 by AuNPs is minimized because long-range electron transfer between the electrode and the AuNPs bound to MBs is not feasible. The high signal amplification and low background current enable the detection of 1 fM target DNA.

Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical–Chemical–Chemical Redox Cycling for the Detection of E. coli 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(NH(3))(6)(3+) 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 10(3) to 10(8) colony-forming units/mL.

Fast catalytic and electrocatalytic oxidation of sodium borohydride on palladium nanoparticles and its application to ultrasensitive DNA detection

We report an ultrasensitive DNA sensor using the rapid enhancement of electrocatalytic activity of DNA-conjugated Pd nanoparticles (NPs); the rapid enhancement results from the fast catalytic hydrolysis of NaBH(4) on Pd NPs and subsequent fast hydrogen sorption into Pd NPs.

Effect of Thermal Treatment on the Electrocatalytic Activities and Surface Roughness of ITO Electrodes

ABSTRACT: The electrocatalytic activities and surface roughness of indium-tin-oxide (ITO) electrodes havebeen investigated after thermal treatment at 100, 150, or 200 o C for 30 min, 2 h, or 8 h. To checkelectrocatalytic activities, the electrochemical behavior of four electroactive species (p-hydro-quinone, Ru(NH 3 ) 63+ , ferrocenemethanol, and Fe(CN) 64– ) has been measured. The electron transferrate for p-hydroquinone oxidation and ferrocenemethanol oxidation increases with increasing theincubation temperature and the incubation period of time, but the rate for Ru(NH 3 ) 63+ is similar irre-spective of the incubation temperature and period because Ru(NH 3 ) 63+ undergoes a fast outer-spherereaction. Overall, the electrocatalytic activities of ITO electrodes increase with increasing the incu-bation temperature and period. The surface roughness of ITO electrodes increases with increasingthe incubation temperature, and the thermal treatment generates many towering pillars as high asseveral tens of nanometer. Keywords : Indium-tin-oxide electrode, Thermal treatment, Electrocatalytic activity, p-hydro-quinone, Ru(NH

Variation of Nitrogen Removal Efficiency and Microbial Communities Depending on Operating Conditions of a CANON Process

Nitrogen removal is one of the most important issues about wastewater treatment because nitrogen is a primary pollutant caused various problems such as eutrophication. We developed a CANON microbial community by using AOB and ANAMMOX bacteria as seeding sources. When 100 mg-N/L of influent ammonium was supplied, the DO above 0.4 mg/L showed a very low TN removal efficiency while the DO of 0.3 mg/L showed TN removal efficiency as high as 71.3%. When the influent ammonium concentration was reduced to 50 mg/L, TN removal efficiency drastically deceased. However, TN removal efficiency was recovered to above 70% after 14 day operation when the influent nitrogen concentration was changed again from 50 mg-N/L to 100 mg-N/L. According to the operating temperature from 37±1°C to 20±1°C, TN removal efficiency also rapidly decreased but gradually increased again up to 70.0±2.6%. The analysis of PCR-DGGE showed no substantial difference in microbial community structures under different operational conditions. This suggests that if CANON sludge is once successfully developed from a mixture of AOB and ANAMMOX bacteria, the microbial community can be stably maintained regardless of the changes in operational conditions.