Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement.

This work describes the development of an electrochemical immunosensor for the analysis of atrazine using recombinant single-chain antibody (scAb) fragments. The sensors are based on carbon paste screen-printed electrodes incorporating the conducting polymer polyaniline (PANI)/poly(vinylsulphonic acid) (PVSA), which enables direct mediatorless coupling to take place between the redox centres of antigen-labelled horseradish peroxidase (HRP) and the electrode surface. Competitive immunoassays can be performed in real-time using this separation-free system. Analytical measurements based on the pseudo-linear relationship between the slope of a real-time amperometric signal and the concentration of analyte, yield a novel immunosensor set-up capable of regenerationless amperometric analysis. Multiple, sequential measurements of standards and samples can be performed on a single scAb-modified surface in a matter of minutes. No separation of bound and unbound species was necessary prior to detection. The system is capable of measuring atrazine to a detection limit of 0.1 ppb (0.1 μg l[-1]). This system offers the potential for rapid, cost-effective immunosensing for the analysis of samples of environmental, medical and pharmaceutical significance

Optimisation and characterisation of biosensors based on polyaniline

With lower limits of detection and increased stability constantly being demanded of biosensor devices, characterisation of the constituent layers that make up the sensor has become unavoidable, since this is inextricably linked with its performance. This work describe the optimisation and characterisation of two aspects of sensor performance: a conductive polymer layer (polyaniline) and the immobilised protein layer. The influence of the thickness of polyaniline films deposited electrochemically onto screen-printed electrode surfaces is described in this work in terms of its influence on a variety of amperometric sensor performance characteristics: time to reach steady state, charging current, catalytic current, background current and signal/background ratios. The influence of polymer film thickness on the conductivity and morphology of finished films is also presented. An electrostatic method of protein immobilisation is used in this work and scanning electron microscopy in conjunction with gold-labelled antibodies and back-scattered electron detection has enabled the direct visualisation of individual groups of proteins on the sensor surface. Such information can provide an insight into the performance of sensors under influence of increasing protein concentrations

Amperometric separation-free immunosensor for real-time environmental monitoring

Immunoanalytical techniques have found widespread use due to the characteristics of specificity and wide applicability for many analytes, from large polymer antigens, to simple haptens, and even single atoms. Electrochemical sensors offer benefits of technical simplicity, speed and convenience via direct transduction to electronic equipment. Together, these two systems offer the possibility of a convenient, ubiquitous assay technique with high selectivity. However, they are still not widely used, mainly due to the complexity of the associated immunoassay methodologies. A separation-free immunoanalytical technique is described here, which has allowed for the analysis of atrazine in real time and in both quasi-equilibrium and stirred batch configurations. It illustrated that determinations as low as 0.13mM (28 ppb) could be made using equilibrium incubation with an analytical range of 0.1–10mM. Measurements could be made between 1 and 10 mM within several minutes using a real-time, stirred batch method. This system offers the potential for fast, simple, cost-effective biosensors for the analysis of many substances of environmental, biomedical and pharmaceutical concern

Decreases in markers of monocyte/macrophage activation after hepatitis C eradication in HIV/HCV co-infected women

OBJECTIVE: Eradication of hepatitis C virus (HCV) in HIV disease decreases liver and non-liver-related morbidity and mortality. Elevated markers of monocyte/macrophage activation (soluble CD163 and sCD14) are associated with excess non-AIDS morbidity and mortality in HIV. We examined the effect of HCV eradication on these markers in relation to change in hepatic fibrosis. DESIGN: Nested substudy within a longitudinal observational cohort METHODS: We studied 126 HIV/HCV coinfected women successfully treated for HCV, with undetectable HCV RNA at least 12 weeks after therapy completion. sCD163 and sCD14 were measured in serum collected before and after HCV eradication. Results were correlated with changes in markers of hepatic fibrosis. RESULTS: Mean age of participants was 56.3 years, mean CD4 was 615, 72% had suppressed HIV RNA. After treatment, sCD163 and sCD14 levels significantly decreased from pre-treatment levels in unadjusted analyses. After adjusting for age, race, hepatic fibrosis status, baseline HCV RNA, CD4 count and HIV RNA status, cigarette smoking, and alcohol use, the decreases in sCD163 and sCD14 remained significant. Decrease in pre-treatment to post-treatment sCD163 were significantly positively correlated with changes in FIB-4 (r=.250, p=.005) and APRI (r=.262, p=.003); similarly decrease in sCD14 was significantly positively correlated with changes in FIB-4 (r=.333, p=.0001) and APRI (r=.457, p<.0001). CONCLUSIONS: HCV eradication is associated with significant reductions in monocyte/macrophage activation markers that correlate with reductions in markers of hepatic fibrosis. These findings support broad access to and early initiation of HCV treatment in order to decrease immune activation and improve health in HIV-infected persons