Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications

The design of an electrochemical glucose sensing device formed by the electrodeposition of multifunctional Au nanoparticles is reported here as a novel concept for an enhanced generic sensing platform. Initially gold nanoparticles (Au) were alternatively coated with a layer of positively charged redox polymer (ORP) and a negatively charged glucose oxidase (GOX) layer alternatively using layer-by-layer methodology to form multifunctional Au/ORP/GOX/ORP particles. The modification and stability of the Au nanoparticles was monitored by using UV-vis spectroscopy and zeta-potential measurements. The modified Au nanoparticles were electrophoretically deposited onto an electrode to produce an electrochemical glucose sensing device. A considerable influence of electrophoretic deposition time and potential was found on the sensing platform response. Preliminary responses to glucose addition showed an enhanced performance by applying an electrophoretic deposition potential of +1.2V vs. Ag/AgCl for 30min. The observed response in the case of microelectrode geometry was in the range of mAcm(2). This work also shows that the presence of a second outer ORP layer on the functionalised Au nanoparticles improved the response.     

DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection

We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.     

Aptamers as elements of bioelectronic devices

A ferrocene labelled aptamer is used as a redox partner of co-immobilised microperoxidase demonstrating a reversible amperometric biomolecular device that could respond to external stimuli in real time.     

Electrochemical immunosensor detection of antigliadin antibodies from real human serum

The determination of antigliadin antibodies from human serum samples is of vital importance for the diagnosis of an autoimmune disease such as celiac disease. An electrochemical immunosensor that mimics traditional ELISA type architecture has been constructed for the detection of antigliadin antibodies with control over the orientation and packing of gliadin antigen molecules on the surface of gold electrodes. The orientation of the antigen on the surface has been achieved using a carboxylic-ended bipodal alkanethiol that is covalently linked with amino groups of the antigen protein. The bipodal thiol presents a long poly(ethyleneglycol)-modified chain that acts as an excellent non-specific adsorption barrier. The bipodal nature of the thiol ensured a good spacing and hence good diffusion properties of electroactive species through the self-assembled monolayer, which is vital for the efficiency of the constructed electrochemical immunosensor. The electrochemical immunosensor was characterized using surface plasmon resonance as well as electrochemical impedance spectroscopy. Amperometric evaluation of the sensor with polyclonal antigliadin antibodies showed stable and reproducible low limits of detection (46 ng/mL; % RSD = 8.2, n = 5). The behaviour and performance of the electrochemical immunosensor with more complex matrixes such as reference serum solutions and real patient samples was evaluated and compared with commercial ELISA kits demonstrating an excellent degree of correlation in thirty minutes total assay time; the electrochemical immunosensor not only delivers a positive or negative result, it allows the estimation of semi-quantitative antibody contents based on the comparison against clinical reference solutions.     

Electrochemical immunosensor for the detection of atrazine

An amperometric immunosensor for the detection of the herbicide atrazine has been developed. A redox polymer PVPOs(bpy)2Cl was co-immobilized with the specific antibody on the surface of the electrode by crosslinking with PEGDGE to form an electron-conducting hydrogel. In a competitive assay the occurrence of the antibody-antigen reaction on the surface of the sensing film was detected through the 'electrical wiring' of the redox centres of antigen-labelled horseradish peroxidase and the electrode surface in the presence of H2O2 at 0.1 V (vsAg/AgCl).     

A multianalyte flow electrochemical cell: application to the simultaneous determination of carbohydrates based on bioelectrocatalytic detection

A multianalyte flow electrochemical cell (MAFEC) for bioanalysis is constructed, characterised and used for simultaneous carbohydrate analysis incorporating mediated amperometric enzyme electrodes. Although multidetection schemes can be addressed with microfabricated systems, it is demonstrated that a "meso" analytical device of low cost can give answers to traditional simultaneous multianalysis problems, being robust, and easy to construct and operate. The cell operates as a radial flow thin-layer device and can achieve mass transport controlled response for fast electrochemical reactions. When appropriate enzymatic electrodes are used the response becomes kinetically limited, but still shows a better than 5% R.S.D. for response to different sugars analysed. All the enzymatic sensors are mediated with different osmium compounds appropriate for each enzyme's mechanism (NAD or PQQ dehydrogenases) in some cases combining multienzyme sensors. All sensors were optimised so that different sugars do not produce interferences to other sensors. Matrix interferences were kept low by operating all sensors at or below 150 mV versus Ag/AgCl. The integrated system was used for the simultaneous detection of fructose, sucrose, glucose, galactose, and lactose, fully characterising the system for these analytes (sensitivity, dynamic range). Cross referenced calibration curves were used for signal treatment and interpretation and it was possible to analyse real juice and milk samples with results agreeing with the standard enzymatic methods for the same analyses with a sampling frequency of more than 100 h(-1).     

Extracellular location of a β-lactamase produced by Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus strains 69 V and CCM 5593 formed a -lactamase that was predominantly found extracellularly in the culture medium. The enzyme has the characteristics of an inducible chromosomally mediated cephalosporinase. Neither cell lysis nor periplasmic leakage are responsible for the extracellular location. The mechanism of secretion is not known.Offprint requests to: P. Borneleit     

Amperometric flow-injection determination of sucrose with a mediated tri-enzyme electrode based on sucrose phosphorylase and electrocatalytic oxidation of NADH

A new sucrose electrode is described for the determination of sucrose without interference from glucose or fructose. The sucrose electrode is based on the tri-enzymatic system of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase, where NAD(P)H is produced from the last enzymatic reaction and recycled into NAD(P)+ through its electrocatalytic oxidation by Os(4,4'-dimethyl-2,2-bypyridine)2(1,10-phenanthroline-5,6-dione). The electrodes were optimised with respect to the various construction parameters and carrier composition in a FIA system and their response as a function of the pH and flow-rate was examined. The electrodes were suitable for operation in a FIA system and the analysis of real samples showed good agreement with the reference method. Typical optimised electrodes showed detection limits of 1 mM sucrose, response time of 5 min, sensitivity 1.010 nA mM(-1), and current density of 8.38 microA cm(-2), using 200 mM PIPES pH 7.25 with 10 mM phosphate and 5 mM MgCl2 as carrier.