Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs.∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.

Identification of dipole disorder in low temperature solution processed oxides: its utility and suppression for transparent high performance solution-processed hybrid electronics.

The ability to deposit high-quality inorganic semiconductors and dielectrics from solution at low process temperatures (∼200 °C) has become a very important research focus. During the course of our investigation, we identify the presence of an induced dipole present in solid state solution processed inorganic oxide insulator layers processed at reduced temperature (200-350 °C) from either molecular precursors, or well-dispersed metal oxide nanoparticles. Chemical composition analysis coupled with electrical measurements shows that the dielectric instability occurs due to proton migration via the Grotthuss mechanism inducing a long lived dipole disorder. Thus we established conditions for suppressing this effect to afford "ideal" high-k dielectric layer. Using this methodology, solution processed all inorganic thin film transistors (TFTs) with charge carrier mobilities exceeding 6 cm2 V-1 s-1 operating at low voltage (5 V) have been achieved. In addition, we show the broad utility of the perovskite high-k dielectric when processed with state of the art polymer and single crystal organic semiconductors yielding mobilities of approx. 7 cm2 V-1 s-1 at only 4 V. These transparent devices demonstrate excellent electrical device stability and a threshold voltage shift of only 0.41 V over 14 h, which is comparable, or better than sputtered oxide films.

How international medical graduates view their learning needs for UK GP training.

International medical graduates (IMGs) form a vital group of general practitioners (GPs) in the NHS. They are known to face additional challenges above and beyond those faced by UK medical graduates in the course of their GP training. Whilst they are a heterogeneous group of professionals, their views on what they need to learn, and how they are supported, are often distant from those of the educators responsible for planning their education. This study was undertaken, through narrative-based focus groups, to explore the issues which matter to the IMGs, in an attempt to empower their voices about their experiences in GP training, and to see what lessons could be drawn from these views. The findings confirmed the central importance, and considerable challenge involved, in making an effective transition into the culture of the NHS and UK general practice. The IMGs felt that induction needed to be an on-going, iterative process of learning which continued throughout training, with a more effective individualised learning needs analysis at the start of GP training. Lack of sophisticated language skills was highlighted as a real concern. Recognition that their lack of knowledge about the NHS at the start of training should not be seen as an indicator of deficiency, but a clue to what they needed to learn were also key messages. IMGs also felt the earlier in their training they undertook a GP placement, the quicker they would start to understand the culture of general practice in the UK. Further work following on from this research should include how to manage change in the educational network for these barriers to be overcome.

A molecular imprinted polymer based sensor for measuring phosphate in wastewater samples.

Phosphate detection in water samples is still completed using colorimetric standard methods, which have a number of disadvantages, to such as being time consuming, requiring filtration, a number of different reagents, frequent calibration and proper disposal of waste chemicals generated. Hence, a simple cost effective analytical method and instrumentation is highly desirable to aid the optimisation of treatment processes and assist the water industry in their efforts to comply with stringent regulations such as the EU's Water Framework Directive. A sensor based on molecular imprinted polymer (MIP) and a conductance transducer was developed for direct and label-free detection of phosphate in water. The sensor was able to measure the presence of phosphate in wastewater samples with good reproducibility, a linear range of 0.66-8 mg P L(-1) and a lower detection limit of 0.16 mg P L(-1). The sensor was further tested to measure phosphate concentrations in unfiltered field samples such as domestic wastewater treatment influent and river water and demonstrated a close correlation with reference measurements. The phosphate MIP sensor offers a way forward as either a handheld sensor for use in the field, or as a potential solution for remote, continuous monitoring of phosphate.

Conductance based sensing and analysis of soluble phosphates in wastewater.

The current standard method used for measuring soluble phosphate in environmental water samples is based on a colourimetric approach, developed in the early 1960s. In order to provide an alternative, label free sensing solution, a molecularly imprinted polymer (MIP) was designed to function as a phosphate receptor. A combination of functional monomer (N-allylthiourea), cross-linker and monomer/template ratios were optimised in order to maximise the binding capacity for phosphate. When produced in membrane format, the MIP's ability to produce a reversible change in conductance in the presence of phosphate was explored for fabrication of a sensor which was able to selectively detect the presence of phosphate compared to sulphate, nitrate and chloride. In wastewater samples the sensor had a limit of detection of 0.16 mg P/l, and a linear range between 0.66 and 8 mg P/l. This is below the minimum monitoring level (1 mg P/l) as required by current legislation for wastewater discharges, making the sensor as developed promising for direct quantification of phosphate in environmental monitoring applications.

Sensing and analysis of soluble phosphates in environmental samples: a review.

Excess phosphate levels in water can lead to increased algal growth, eutrophication and reduced water quality. Phosphate levels in water are regulated by the EU through the Urban Waste Water Treatment Directive (annual mean total phosphorus concentrations of 1-2 mg/l) and the Water Framework Directive that will enforce "good ecological and chemical status" by 2015. Legislation is therefore driving the need for increased monitoring of soluble phosphate in water, escalating the desire for a direct, label free approach that could provide remote, continuous monitoring in real-time. The standard method for measuring soluble phosphate in water is a colourimetric technique developed in the 1960s. This colourimetric approach is difficult to adapt for on-line measurements, uses specific reagents which require safe disposal and thus incurs significant costs to the water industry when carried out on a large scale. This review considers optical and electrochemical sensors plus recent advances with synthetic receptors and molecularly imprinted polymers. Progress in the development of phosphate sensors, designed for use in a variety of disciplines, is highlighted with a view to adapting successful approaches for use in the water sector. Additional considerations include the need for long term stability, low maintenance, specificity for phosphate and the capability of measuring total phosphorus concentrations down to at least 1 mg/l, as required by legislation. A sensor that could directly measure soluble, inorganic phosphate concentrations would draw significant interest from the environment sector and other disciplines, including the agricultural, detergent and bio-medical industries.