We previously reported a colourimetric assay

We previously reported a colourimetric assay based on a SNAP-25 monolayer on colloidal gold [8]. This assay has been developed into an electrochemical assay presented in this paper. Electrochemical techniques, such as cyclic voltammetry and electrochemical impedance spectroscopy, are label-free techniques that are highly sensitive to changes and interactions at a surface. Over the past few years a number of advances have been made in this area with the development of sensors specific for various toxins, pathogens and biomarkers [3,12,17] due to their high sensitivity and rapid detection times. Cyclic voltammetry provides information on the amount of oxidisable buy zip on a working electrode and the number of electrons involved in the oxidation, which is given in terms of charge [13]. Variations in the charge indicate binding events and changes to the self-assembled monolayer. Impedance is a measure of the complex resistance met when current flows through a circuit made up of resistors, capacitors and inductors. Electrochemical impedance spectroscopy utilises redox probes such as Fe(CN)63−/4− or Ru(NH3)62+/3+ measuring the ability of these ions to become oxidised and reduced at the working electrode [1]. At unmodified working electrodes the route to the electrode surface is not blocked and these chemicals easily undergo the reactions. If the electrode surface is modified by an alkanethiol or a protein then the ions are more blocked increasing the charge transfer resistance (Rct) of the circuit [11].
Materials and methods Gold working electrodes were purchased from Winkler GmbH Germany, SNAP-25 was from Abcam (ab155885) and all other chemicals were purchased from Sigma Aldrich. Dysport® samples containing lyophilised Clostridium botulinum Type A toxin-haemaggluttinin complex (4.35ng), human serum albumin (125μg) and lactose (2.5mg) and placebos containing the excipients from Dysport® were kindly supplied by IPSEN Biopharm. Water was purified and had a nominal resistivity of 18MΩcm at 25°C. All measurements were performed using an Autolab PGSTAT 30 computer-controlled electrochemical measurement system (Eco Chemie, Holland) with a home-made three electrode cell with a SNAP-25 modified Au(111) working electrode, a platinum counter electrode and a saturated calomel reference electrode.
Results and discussion
Conclusion Industrial pressures have caused a demand for quicker, cheaper assays for botulinum neurotoxin. The assays presented in the paper are much quicker than the mouse bioassay taking hours rather than days to perform. The impedance biosensor also outperforms the mouse bioassay on sensitivity producing a response down to 25pg/mL of toxin. The major benefit of these assays over some others in development, such as ELISA, is the detection of active toxin through monitoring the proteolytic activity rather than just the presence of the molecule. These assays also clearly distinguish between the toxin product and the placebo sample which has previously caused problems due to the slight proteolytic activity of HSA [9]. When performed correctly EIS does not damage the biological layer giving an advantage, allowing for direct comparison between the individual SNAP-25 monolayers before and after incubation with the toxin.
Conflict of interest
Acknowledgements The authors would like to thank IPSEN Biopharm and the Knowledge Economy Skills Scholarship (KESS) for funding the initial research and the Welsh European Funding Office for funding the current project. We would also like to thank IPSEN Biopharm for supplying samples of Dysport® and placebo. This work is included in patent application number 1310090.4 [6].
Introduction The genus Trichoderma is a saprophytic fungi which can be found in all climatic zones of the world. The genus Trichoderma was first described in 1794 by Persoon [1] and there are reportedly almost 130 species [2]. The conventional technique used to identify and classify Trichoderma species is based on phenotypic traits which include morphological and biochemical characteristics; however, it is quite difficult to differentiate between very closely related species. DNA sequences can be used for the identification of fungi at the species level, but approximately 40% of the GenBank database sequences of Trichoderma species have been erroneously identified or remain unidentified at the species level [3,4]. There are a large number of sequences deposited in the GenBank that are incorrectly labeled and unless remedied they will continue to be assigned to the wrong taxa [5,6]. Under these conditions, the best conceivable conception of the molecular data and morphological characteristics of isolates is achieved using detailed photographs or drawings of the specimens to prevent any controversial identification at the species level [7]. To easily recognize and ensure the quality of results it is also possible to go back to the main source of the information. Otherwise, species level identification is difficult to do correctly, especially when it is necessary to rely on a source that has made a misidentification.