Eco-friendly voltammetric platform for trace metal determination using a conductive polymer sensor modified with bismuth nanoparticles generated by spark discharge.

The fabrication of a low-cost eco-friendly sensor platform for the voltammetric determination of trace metals by electrochemical stripping analysis is reported. Plastic conductive electrodes were manufactured via injection moulding from polysterene reinforced with carbon fibres. The platform comprises a carbon counter electrode, a working electrode modified with bismuth nanoparticles generated by spark discharge and a reference electrode coated with AgCl. The sensor fabrication and modification procedures are simple, cost-effective and fast while the materials used are environment-friendly. The utility of the voltammetric platform is demonstrated for stripping analysis of Cd(II) and Pb(II); the limits of detection are 0.7 µg L-1 and 0.6 µg L-1, respectively (with a deposition time of 240 s) which are comparable to conventional Bi-modified sensors and are sufficient to determine the target metals in water and food samples. The scope of the analytical platform for multi-element assays and for the determination of other trace metals is discussed with representative examples. Therefore, this sustainable and economical platform holds great potential for electrochemical sensing of trace metals.

Gold nanoparticle-modified sustainable plastic sensor chip for voltammetric monitoring of Hg(II).

Mercury is a toxic environmental contaminant that can cause serious health problems. This work describes a new type of eco-friendly three-electrode plastic sensor chip for the determination of trace Hg(II) by means of anodic stripping voltammetry (ASV). The sensor chip is entirely fabricated by injection moulding, which is a sustainable manufacturing method, and consists of three conductive carbon-based electrodes embedded in a plastic holder while the reference electrode is coated with Ag using e-beam evaporation. The sample is spiked with Au(III) which deposits on the working electrode in the form of gold nanoparicles during the analysis; the target Hg(II) co-deposits on the gold nanoparticles forming a Au(Hg) amalgam in situ. The accumulated Hg is stripped off the electrode and quantified by an anodic square wave potential scan. The relevant conditions and the potential interferences are investigated. The limit of detection for Hg(II) is 0.4 µg L-1 and the repeatability at the 20 µg L-1 Hg(III) level (n = 10) is 5.3%. The sensor is applied to water, honey, fish oil and mussel samples with recoveries between 98 and 107%.

Disposable Injection Molded Conductive Electrodes Modified with Antimony Film for the Electrochemical Determination of Trace Pb(II) and Cd(II).

This work describes a novel electrochemical sensor fabricated by an injection molding process. This device features a conductive polymer electrode encased in a plastic holder and electroplated in situ with a thin antimony film. The antimony film sensor was applied to the determination of Pb(II) and Cd(II) by anodic stripping voltammetry (ASV). The deposition of Sb on the sensor was studied by cyclic voltammetry (CV) and microscopy. The experimental variables (concentration of the antimony plating solution, deposition potential and time, stripping waveform) were investigated, and the potential interferences were studied and addressed. The limits of detection were 0.95 µg L-1 for Pb(II) and 1.3 for Cd(II) (at 240 s of preconcentration) and the within-sensor percentage relative standard deviations were 4.2% and 4.9%, respectively, at the 25 µg L-1 level (n = 8). Finally, the sensor was applied to the determination of Pb(II) and Cd(II) in a phosphorite sample and a lake water sample.

Determination of Pb(II) by sequential injection/stripping analysis at all-plastic electrochemical fluidic cells with integrated composite electrodes.

This work reports the development of a sequential injection/stripping analysis method for the determination of trace Pb(II) at injection-moulded electrochemical fluidic cells. Conducting carbon fibre-loaded polystyrene electrodes were integrated within the plastic cells. The flow-through cells were incorporated into a home-made sequential injection analysis (SIA) manifold. Different experimental parameters for the detection of Pb(II) were investigated including the type and concentration of the supporting electrolyte, the conditions of the stripping step, the volume of the sample, the flow rate and the accumulation potential. The LOD for Pb(II) was 0.5µgL(-1), the within-cell % relative standard deviation (n=8) was 3.1% and the between-cell % relative standard deviation (n=5) was 8.9% for 25µgL(-1) Pb(II). The cells were applied to the determination of Pb(II) in tapwater and a phosphate fertilizer sample.

Scaling-up ultrasound standing wave enhanced sedimentation filters.

Particle concentration and filtration is a key stage in a wide range of processing industries and also one that can be present challenges for high throughput, continuous operation. Here we demonstrate some features which increase the efficiency of ultrasound enhanced sedimentation and could enable the technology the potential to be scaled up. In this work, 20 mm piezoelectric plates were used to drive 100 mm high chambers formed from single structural elements. The coherent structural resonances were able to drive particles (yeast cells) in the water to nodes throughout the chamber. Ultrasound enhanced sedimentation was used to demonstrate the efficiency of the system (>99% particle clearance). Sub-wavelength pin protrusions were used for the contacts between the resonant chamber and other elements. The pins provided support and transferred power, replacing glue which is inefficient for power transfer. Filtration energies of ∼4 J/ml of suspension were measured. A calculation of thermal convection indicates that the circulation could disrupt cell alignment in ducts >35 mm high when a 1K temperature gradient is present; we predict higher efficiencies when this maximum height is observed. For the acoustic design, although modelling was minimal before construction, the very simple construction allowed us to form 3D models of the nodal patterns in the fluid and the duct structure. The models were compared with visual observations of particle movement, Chladni figures and scanning laser vibrometer mapping. This demonstrates that nodal planes in the fluid can be controlled by the position of clamping points and that the contacts could be positioned to increase the efficiency and reliability of particle manipulations in standing waves.

Using Fourier transform IR spectroscopy to analyze biological materials.

IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Using droplet-based microfluidic technology to study the precipitation of a poorly water-soluble weakly basic drug upon a pH-shift.

The purpose of this study is to develop a droplet-based microfluidic device capable of monitoring drug precipitation upon a shift from gastric pH (pH 1.5) to intestinal pH (pH 6.5-7.0). The extent of precipitation occurring in droplets over time was measured using a novel on-chip laser scattering technique specifically developed for this study. The precipitation of ketoconazole, a poorly water-soluble basic drug, was investigated under different concentrations and pH values. It has been shown that the drug precipitates rapidly under supersaturation. Two water-soluble aqueous polymers, namely, polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) have been evaluated as precipitation inhibitors. HPMC was shown to be the most potent precipitation inhibitor. It is envisaged that the microfluidic pH-shift method developed in this study would form a proof-of-concept study, towards the development of a high throughput method for screening pharmaceutical excipients/precipitation inhibitors.

Separation of alkali metals using isotachophoresis with cryptand 222 as a leading electrolyte additive.

This work shows how the inclusion of cryptand 222 as a leading electrolyte additive in isotachophoresis affects the electrophoretic mobilities of alkali metal cations. Using isotachophoresis the separation of alkali metals can be difficult due to the similar electrophoretic mobilities of three of the ions: caesium, rubidium and potassium. However, the presence of cryptand 222 in the leading electrolyte retards the mobilities of the caesium, rubidium and potassium to a different extent allowing separations to be made. A novel electrolyte system was formulated which consisted of a leading electrolyte of 10 mM caesium hydroxide, 0.75 mM cryptand 222 buffered to pH 9.4 with glycylglycine and a terminating electrolyte of 10 mM tetrabutylammonium hydroxide. The use of this electrolyte system allowed good separations of mixtures of rubidium, potassium, sodium and lithium to be achieved. The method was also applied to the analysis of edible salt samples.

Miniaturised free flow isotachophoresis of bacteria using an injection moulded separation device.

A new design of miniaturised free flow electrophoresis device has been produced. The design contains a separation chamber that is 45 mm long by 31.7 mm wide with a depth of 50 µm and has nine inlet and nine outlet holes to allow for fraction collection. The devices were formed of polystyrene with carbon fibre loaded polystyrene drive electrodes and produced using injection moulding. This means that the devices are low cost and can potentially be mass produced. The devices were used for free flow isotachophoresis (FFITP), a technique that can be used for focussing and concentrating analytes contained within complex sample matrices. The operation of the devices was demonstrated by performing separations of dyes and bacterial samples. Analysis of the output from FFITP separations of samples containing the bacterium Erwinia herbicola, a biological pathogen, by cell culturing and counting showed that fractionation of the output was achieved.

Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography.

We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and ß-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.

A microfluidic device for self-synchronised production of droplets.

The primary requirement for a mixing operation in droplet-based microfluidic devices is an accurate pairing of droplets of reaction fluids over an extended period of time. In this paper, a novel device for self-synchronous production of droplets has been demonstrated. The device uses a change in impedance across a pair of electrodes introduced due to the passage of a pre-formed droplet to generate a second droplet at a second pair of electrodes. The device was characterised using image analysis. Droplets with a volume of ~23.5 ± 3.1 nl (i.e.~93% of the volume of pre-formed droplets) were produced on applying a voltage of 500 V. The synchronisation efficiency of the device was 83%. As the device enables self-synchronised production of droplets, it has a potential to increase the reliability and robustness of mixing operations in droplet-based microfluidic devices.

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase.

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.

Thiocyanate and nitrite analysis using miniaturised isotachophoresis on a planar polymer chip.

A new method has been developed to improve the determination of thiocyanate using isotachophoresis. This method uses complexation with copper(II) as a mechanism for improving the separation of thiocyanate from chlorate and perchlorate. By using a pH of 3.25 the method can also be used to analyse nitrite. Separations were carried out using a miniaturised poly(methyl methacrylate) (PMMA) separation device. Linearity was observed from 1.25 to 75 mg dm(-3) with a correlation coefficient of 0.998 for both thiocyanate and nitrite. Limits of detection for these two species were calculated to be 0.8 mg dm(-3) and 0.9 mg dm(-3) respectively. The method was successfully applied to the analysis of these anions in a range of samples including explosive residues.

A specific, robust, and automated method for routine at-line monitoring of the concentration of cellulases in genetically modified sugarcane plants.

Bagasse is one of the waste crop materials highlighted as commercially viable for cellulosic bio-ethanol production via enzymatic conversion to release fermentable sugars. Genetically modified sugarcane expressing cellobiohydrolases (CBH), endoglucanase (EG), and ß-glucosidases (BG) provide a more cost-effective route to cellulose breakdown compared to culturing these enzymes in microbial tanks. Hence, process monitoring of the concentration profile of these key cellulases in incoming batches of sugarcane is required for fiscal measures and bio-ethanol process control. The existing methods due to their non-specificity, requirement of trained analysts, low sample throughput, and low amenability to automation are unsuitable for this purpose. Therefore, this paper explores a membrane-based sample preparation method coupled to capillary zone electrophoresis (CZE) to quantify these enzymes. The maximum enzyme extraction efficiency was obtained by using a polyethersulfone membrane with molecular cut-off of 10 kDa. The use of 15 mM, pH 7.75, phosphate buffer resulted in CZE separation and quantification of CBH, EG, and BG within 10 min. Migration time reproducibility was between 0.56% and 0.7% and hence, suitable for use with automatic peak detection software. Therefore, the developed CZE method is suitable for at-line analysis of BG, CBH, and EG in every batch of harvested sugarcane.

Determination of the potassium content of explosive residues using miniaturised isotachophoresis.

A new method has been developed to allow the determination of potassium in post-explosion residues to be made using miniaturised isotachophoresis. The method is based on the use of a caesium leading ion with 4.5 mM 18-crown-6 ether added to retard the potassium to allow reliable determinations to be made. With the conditions selected no interference was noted from other small inorganic cations, such as ammonium, barium, calcium, magnesium, sodium or strontium. The method was successfully applied to the analysis of seven samples containing explosive residues obtained from the unconfined burning of several flash powders. The procedure was found to offer good linearity for potassium determinations over the concentration range of 1.25-150 µg/mL with a coefficient of determination of 0.999 achieved.

Isotachophoresis-based sample preparation of cellulases in sugarcane juice using bovine serum albumin as a model protein.

The foremost requirement of quantification of cellulases expressed in genetically modified sugarcane is an efficient sample clean-up. This work investigates the feasibility of isotachophoresis for this purpose. An electrolyte system comprising a leading electrolyte of 10mM formic acid at pH 9.0 and a terminating electrolyte of 10mM ß-alanine was devised and used to perform isotachophoresis of cellulases. The use of a simple front cutting method removed a majority of interfering species in the juice, thereby resulting in the formation of a distinct zone of desired proteins. In comparison to techniques such as ultrafiltration and liming, the analysis time and loss of desired proteins was lower when the sample was prepared by using isotachophoresis. Hence, isotachophoresis was an ideal choice for purification of the proteins in question from the remaining components in the juice.

Continuous two-phase flow miniaturised bioreactor for monitoring anaerobic biocatalysis by pentaerythritol tetranitrate reductase.

A novel continuous recirculating two-phase flow miniaturised bioreactor was developed for biocatalytic transformations with the enzyme pentaerythritol tetranitrate reductase using on-chip spectroscopic detection of the organic and aqueous phases. A phase separation technique is described that uses electrostatic attraction to force charged droplets to merge back into the aqueous phase and thus allow the monitoring of both reaction phases during enzymatic turnover. We report an increased rate of enzyme catalysed reduction of trans-2-(2-nitrovinyl)thiophene, which was used as a model system to demonstrate the principles of the bioreactor design, compared to conventional macroscale experiments. Additional data obtained with ketoisophorone, trans-cinnamaldehyde and 2-methylmaleimide support our findings and provide a basis for improving the chemistry of biocatalysis.

Optimisation of secondary electrospray ionisation (SESI) for the trace determination of gas-phase volatile organic compounds.

An electrospray ionisation triple quadrupole mass spectrometer (Varian 1200 L) was modified to accept nitrogen samples containing low concentrations of volatile organic compounds. Six candidate probe compounds, methyl decanoate, octan-3-one, 2-ethylhexanoic acid, 1,4-diaminobutane, dimethyl methylphosphonate, and 2,3-butanediol, at concentrations below 50 ppb(v) were generated with permeation tubes in a test atmosphere generator. The concept of using a set of molecular probes to evaluate gas-phase electrospray ionisation of volatile analytes was assessed and the feasibility of adopting a unified ionisation approach for gas and liquid contamination of exobiotic environments established. 450 experiments were run in a five-replicate, fifteen-level, three-factor, central-composite-design with exponential dilution for each of the six probe compounds studied. The three factors studied were ionisation voltage, drying-gas flow and nebulising-gas flow. Parametric modelling by regression analysis enabled the differences in the ionisation behaviours of the probe compounds to be described by the optimisation models. Regression coefficients were in the range 0.91 to 0.99, indicating satisfactory levels of precision in the optimisation models. A wide range in ionisation efficiency was observed, with different optimised conditions required for the probe compounds. It was evident that no one factor appeared to dominate the response and the different factors produced different effects on the responses for the different molecules. 1,4-Butanediamine and dimethyl methylphosphonate required significantly lower ionisation voltages (1.2 kV) than the other four, which achieved optimised sensitivity towards the maximum voltage used in this design (5 to 6 kV). Drying-gas flow rates were found to be more important than nebulising-gas flow rates. However, variations in the constant term B(0) in the optimisation models indicated that other factors, not included in this study, were also likely to be involved in the ionisation process. Electrolyte-flow rate and ionisation temperature were proposed for follow up studies. Exponential dilution data indicated sensitive and analytically useful responses in the target range of 5 to 50 ppb(v) for all six compounds. Significantly, responses were seen at concentrations significantly below 5 ppb(v), with sub ppt(v) responses observed for 1,4-butanediamine, 2-ethylhexanoic acid, dimethylmethylphosphonate, and 1,3-butanediol. Responses in the ppt(v) to ppb(v) range were observed for the remaining two compounds. The observations from this study demonstrated the utility of adopting a set of probe compounds to evaluate electrospray ionisation performance for volatile organic compound based assays; indicated the existence of multiple ionisation mechanisms; and revealed potential sensitivity at the parts per quadrillion level ppq(v).

Precision milled flow-cells for chemiluminescence detection.

Novel flow-cells with integrated confluence points and reaction channels designed for efficient mixing of fast chemiluminescence systems were constructed by machining opposing sides of a polymer chip and sealing the channels with transparent epoxy-acetate films. A hole drilled through the chip provided the conduit from the confluence point on one side to the centre of the reaction zone on the other side, allowing rapid presentation of the reacting mixture to the photodetector. The effectiveness of each flow-cell was evaluated by comparing the chemiluminescence intensity using flow injection analysis methodology, and examining the distribution of light emanating from the reaction zone (captured by photography in a dark room) when the reactants were continuously merged. Although previously reported chemiluminescence detectors constructed by machining channels into polymers have almost exclusively been prepared using transparent materials, we obtained far greater emission intensities using an opaque white chip with a thin transparent seal, which minimised the loss of light through surfaces not exposed to the photomultiplier tube. Furthermore, this approach enabled the exploration of reactor designs that could not be incorporated in traditional coiled-tubing flow-cells.

Toward a microfluidic-based rapid amylase assay system.

This article describes work into a prototype system for the assay of amylase, using microfludic technologies. The new system has a significantly shorter cycle time than the current laboratory methods, which generally use microtitre plates, yet is capable of generating significantly superior results. As such, we have shown that sensitivity is enhanced by a factor of 10 in the standard assay trials, and by a factor of 2 in the real-sample lab trials. In both assays, the use of a microreactor system reduced the reaction time by a factor of 6.2, from 20 min incubation to 3.2 min. Basing the conclusion on the Megazyme Cerealpha Standard Method, and using the Cerealpha units as a measure of assay efficiency, the typical response for the microfluidic assay was shown to be 1.0 x 10(-3) CU/mL (standard deviation [SD] 2.5 x 10(-4) CU/mL), compared to 2.56 x 10(-4) CU/mL (SD 5.94 x 10(-5) CU/mL) for the standard macroassay. It is believed that this improvement in the reaction schematics is due to the inherent advantages of microfluidic devices such as superior mixing, higher thermal efficiency, and enhanced reaction kinetics.