Paul A. Millner

Arabidopsis AUX1 gene: A permease-like regulator of root gravitropism

The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.

Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Biosensors for Whole-Cell Bacterial Detection

SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost.

The effect of heavy metals accumulation on the chlorophyll concentration of Typha latifolia plants, growing in a substrate containing sewage sludge compost and watered with metaliferus water

Typha latifolia plants, commonly known as cattails, were grown in a mixture of sewage sludge compost, commercial compost and perlite. Four groups (A, B, C and D) were irrigated (once every 2 weeks) with a solution containing different concentrations of Cd, Cu, Ni, Pb and Zn, where in the fifth (group M) tap water was used. At the end of the 10 weeks experimental period the mean concentration of Ni, Cu and Zn in the roots and leaves of the plants in the four groups was significantly larger to that of the plants of group M. A linear regression test satisfactorily correlated the metals’ concentrations in the irrigation solutions with the metals concentration in the leaves and roots of groups A, B, C and D. The concentration of total chlorophyll, chlorophyll a (chla) and chlorophyll b (chlb) in the leaves of the developing plants was also monitored in 2 weeks intervals. Groups A, B, C and M presented an increasing concentration of total chlorophyll, with time. In group D (stronger solution), the mean total chlorophyll concentration was reduced from 1080.69 mg/g fresh weight (f.w.) in the 8th week to 715.14 mg/g f.w., in the 10th week, a probable evidence of inhibition. When statistically tested, it was suggested that there was no significant difference between the mean chlorophyll values of the groups in each set of samples, concluding that no significant toxic action was imposed in the plants by the metals. However, when similar statistical analysis was implemented in the ratios of chla and chlb, there was significant reduction of the ratios in groups D plants, suggesting some increase in chlorophyll hydrolysis due to the metals accumulation (toxic effect) in comparison with the other groups. # 2003 Elsevier Science B.V. All rights reserved.

A review on viral biosensors to detect human pathogens

Rapid identification of viruses has important implications for medical healthcare. Current methods for identification and quantification of particular virus are time consuming and often expensive. Therefore, demand for sensitive and accurate viral biosensors with rapid detection systems is increasing. A hand held biosensing device would give fast, reliable results for identifying and quantitating the number of virus particles in a sample. Techniques currently being applied to achieve this aim include electrochemical biosensors, based on amperometric, potentiometric and impedance measurement, optical biosensors using surface plasmon resonance (SPR), optical fibers and piezoelectric biosensors based on microcantilevers. Future research also looks to the use of nanoparticles and novel nanomaterials as alternate recognition surfaces for use in a variety of sensor formats.

A label-free electrical impedimetric biosensor for the specific detection of Alzheimer's amyloid-beta oligomers

Alzheimers disease (AD) is the most common form of dementia, with over 37 million sufferers worldwide and a global cost of over

Biosensor Regeneration: A Review of Common Techniques and Outcomes

600 billion. There is currently no cure for AD and no reliable method of diagnosis other than post-mortem brain examination. The development of a point-of-care test for AD is an urgent requirement in order to provide earlier diagnosis and, thus, useful therapeutic intervention. Here, we present a novel, label-free impedimetric biosensor for the specific detection of amyloid-beta oligomers (AβO), which are the primary neurotoxic species in AD. AβO have been proposed as the best biomarker for AD and levels of AβO in the blood have been found to correlate with cerebrospinal fluid load. The biorecognition element of our biosensor is a fragment of the cellular prion protein (PrP(C), residues 95-110), a highly expressed synaptic protein which mediates the neuronal binding and toxicity of AβO. During the layer-by-layer sensor construction, biotinylated PrP(C) (95-110) was attached via a biotin/NeutrAvidin bridge to polymer-functionalised gold screen-printed electrodes. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry and scanning electron microscopy were used to validate biosensor assembly and functionality. EIS was employed for biosensor interrogation in the presence of Aβ oligomers or monomers. The biosensor was specific for the detection of synthetic AβO and gave a linear response, without significant detection of monomeric Aβ, down to an equivalent AβO concentration of ~0.5 pM. The biosensor was also able to detect natural, cell-derived AβO present in conditioned medium. The eventual commercialisation of this biosensor system could allow for the early diagnosis and disease monitoring of AD.

Labeless AC impedimetric antibody-based sensors with pg ml−1 sensitivities for point-of-care biomedical applications

Biosensors are ideally portable, low-cost tools for the rapid detection of pathogens, proteins, and other analytes. The global biosensor market is currently worth over 10 billion dollars annually and is a burgeoning field of interdisciplinary research that is hailed as a potential revolution in consumer, healthcare, and industrial testing. A key barrier to the widespread adoption of biosensors, however, is their cost. Although many systems have been validated in the laboratory setting and biosensors for a range of analytes are proven at the concept level, many have yet to make a strong commercial case for their acceptance. Though it is true with the development of cheaper electrodes, circuits, and components that there is a downward pressure on costs, there is also an emerging trend toward the development of multianalyte biosensors that is pushing in the other direction. One way to reduce the cost that is suitable for certain systems is to enable their reuse, thus reducing the cost per test. Regenerating biosensors is a technique that can often be used in conjunction with existing systems in order to reduce costs and accelerate the commercialization process. This article discusses the merits and drawbacks of regeneration schemes that have been proven in various biosensor systems and indicates parameters for successful regeneration based on a systematic review of the literature. It also outlines some of the difficulties encountered when considering the role of regeneration at the point of use. A brief meta-analysis has been included in this review to develop a working definition for biosensor regeneration, and using this analysis only ∼60% of the reported studies analyzed were deemed a success. This highlights the variation within the field and the need to normalize regeneration as a standard process across the field by establishing a consensus term.

Label-less Immunosensor Assay for Myelin Basic Protein Based upon an ac Impedance Protocol

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.

Are guanine nucleotide-binding proteins involved in regulation of thylakoid protein kinase activity?

This paper describes the development and characterization of a label-less immunosensor for myelin basic protein (MBP) and its interrogation using an ac impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Polyaniline was electrodeposited onto the sensors, and this modified surface then utilized to immobilize a biotinylated antibody for MBP using a classical avidin-biotin approach. Electrodes containing the antibodies were exposed to solutions of MBP and interrogated using an ac impedance protocol. The real component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a nonspecific IgG antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors, thereby accounting for and eliminating the effects of nonspecific adsorption of MBP. A logarithmic relationship between the concentration of MBP in buffer solutions and the impedimetric response was observed.