Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7.

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.

A sensitive enzyme-linked immunosorbent assay for detecting carcinogenic aristolochic acid in herbal remedies.

Aristolochic acid, a naturally occurring nephrotoxin and rodent carcinogen, has been associated with the development of various nephropathies in humans. Developing a sensitive and rapid method to screen the aristolochic acid levels in herbal remedies is urgent for protecting public health. Polyclonal antibodies for aristolochic acid were generated from rabbits after the animals had been immunized with either aristolochic acid-ovalbumin (OVA) or aristolochic acid-keyhole limpet hemocyanin (KLH). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) and a competitive direct enzyme-linked immunosorbent assay (cdELISA) were used for the characterization of the antibodies and for analysis of aristolochic acid contaminated in herbal medicine and diet pills. The antibody titers in the serum of rabbits immunized with aristolochic acid-OVA were considerably higher than those from aristolochic acid-KLH-immunized rabbits. The antibodies from the aristolochic acid-OVA-immunized rabbits were further characterized. In the ciELISA with aristolochic acid-KLH as the plate-coating antigen, the concentrations of the aristolochic acid mixture, aristolochic acid I, and aristolochic acid II that caused 50% inhibition (IC50) of binding of antibodies to aristolochic acid-KLH were found to be 1.2, 0.7, and 18 ng/mL, respectively. When 0.25-5 microg/g of standard aristolochic acid was spiked to ground lotus seeds and then extracted with 0.01 M phosphate-buffered saline, the recovery rate was found to be 86.5% in the ciELISA. Analysis of aristolochic acid in herbal medicine and diet pills with ciELISA showed that 10 of the 12 examined samples were contaminated at levels from 0.6 to 655 microg/g. The presence of aristolochic acid was also confirmed by the high-performance liquid chromatography method.

Reduction of the interferences of biochemicals and hematocrit ratio on the determination of whole blood glucose using multiple screen-printed carbon electrode test strips.

A practical approach to reduce the interferences of biochemicals and hematocrit ratio (Hct%) in the determination of whole blood glucose using multiple screen-printed carbon electrode (SPCE) test strips is described. SPCE test strips with and without glucose oxidase [i.e., GOD(+)-SPCEs and GOD(-)-SPCEs] were used and the chronoamperometric currents of test glucose solutions with various spiked uric acid concentrations and Hct% were measured. By establishing the interference relationships between glucose concentrations and uric acid concentrations as well as Hct% values and with appropriate corrections, the whole blood glucose determinations could be made to be more accurate and comparable to those determined by the reference YSI method. Specifically, the use of the DeltaI value, i.e., the current difference between GOD(+)-SPCE and GOD(-)-SPCE measurements, would reduce most of the uric acid/biochemical interferences. An interpolation method was also established to correct for the glucose determinations with Hct% interferences. The Hct% corrections using the interpolation method are especially important and necessary for those blood samples with glucose concentrations higher than 110 mg dL(-1) and Hct% values lower than 35%. This approach should also be applicable to other biochemical determinations using similar electrochemical techniques.

Portable electrochemical blood uric acid meter.

We describe a new portable uric acid (UA) meter, called the UASure (Apex Biotechnology Corp., Hsinchu, Taiwan). The UASure is an electrochemical blood UA meter designed for fast monitoring of UA concentrations in one drop of capillary blood using an electrochemical test strip. We compared the UASure with the standard method, the Hitachi 7600 modular system (Hitachi, Tokyo, Japan), in 146 volunteers (average age 62.5 +/- 12.8 years). Of these, 65 were known hyperuricemic subjects, 17 of whom received medical therapy. The patients donated their capillary and venous blood samples in random order. Capillary blood and one drop of venous blood were tested immediately by the UASure. The venous blood in the test tube was sent to the central laboratory for serum UA measurement by the Hitachi 7600. The intra-assay coefficients of variation (CVs) of the UASure were 4.79%, 5.77%, and 3.08% at UA levels of 5.8, 7.1, and 13.5 mg/dl, respectively. The UA concentrations tested by the UASure correlated well with those by the Hitachi 7600 (r = 0.87 in venous sampling and r = 0.78 in capillary sampling, P < 0.001). The intraclass correlation was good for venous samples by the UASure (rI = 0.84, 95% CI 0.82-0.90), somewhat below the meaningful criterion for capillary samples by the UASure (rI = 0.77, 95% CI 0.69-0.83). UASure with venous sampling is interchangeable with the standard method for UA measurement.