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Electrochemical detection of amino acids at carbon nanotube and nickel-carbon nanotube modified electrodes.

The oxidation and enhanced detection of traditionally 'non-electroactive' amino acids at a single-wall carbon nanotube (SWNT) surface and at a nickel hydroxide film electrochemically deposited and generated upon the SWNT layer is reported. Different CNT are compared, with Nafion-dispersed SWNT offering the most favorable layer for constant-potential amperometric detection. Factors affecting the oxidation process, including the pH or applied potential, are assessed. The response of the SWNT-Nafion coated electrode compares favorably with that of copper and nickel disk electrodes under flow injection analysis (FIA) conditions. The electrodeposition of nickel onto the SWNT-Nafion layer (Ni-CNT) led to a dramatic enhancement of the analytical response (vs. that observed at the SWNT or nickel electrodes alone). The oxidative process at the Ni(OH)(2) layer has been studied and the increase in sensitivity rationalized. In the presence of amino acid the Ni-CNT layer undergoes an electrocatalytic process in which the amino acid reduces the newly formed NiO(OH) species. Furthermore, the attractive response of both the CNT and Ni-CNT layers has allowed these electrodes to be used for constant-potential FIA of various amino acids and indicates great promise for monitoring chromatographic effluents. Once again an improved signal was observed at the Ni-CNT electrode compared to nickel deposited upon a bare glassy carbon electrode (Ni-GC).

Amino Acids↗

Electroosmotic mixing in microchannels.

Mixing is an essential, yet challenging, process step for many Lab on a Chip (LOC) applications. This paper presents a method of mixing for microfluidic devices that relies upon electroosmotic flow. In physical tests and in computer simulations, we periodically vary the electric field with time to mix two aqueous solutions. Good mixing is shown to occur when the electroosmotic flow at the two inlets pulse out of phase, the Strouhal number is on the order of 1, and the pulse volumes are on the order of the intersection volume.

Complex Mixtures↗

High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems.

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Antibodies↗

Macro-to-micro interfaces for microfluidic devices.

Since the concept of miniaturized total analysis systems (microTAS) was invented, a great number of microfluidic devices have been demonstrated for a variety of applications. However, an important hurdle that still needs to be cleared is the connection of a microfluidic device with the rest of the world, which is often referred to as the macro-to-micro interface, interconnect, or world-to-chip interface. In this review, we will examine the methods used by pioneers in the field and other investigators, review the approaches for capillary electrophoresis-based devices and those using pneumatic pumping, and present additional discussion on interface standardization and choosing and designing interconnects for your applications.

Electrophoresis, Capillary↗

In-situ quantitative analysis of a prostate-specific antigen (PSA) using a nanomechanical PZT cantilever.

We report on a novel technique of resonant frequency shift measurement based on a nanomechanical cantilever with a PZT actuating layer for label-free detection of a prostate-specific antigen (PSA) in a liquid environment. The nanomechanical PZT thin film cantilever is composed of SiO(2)/Ta/Pt/PZT/Pt/SiO(2) on a SiN(x) supporting layer for simultaneous self-exciting and sensing; it was fabricated using a standard MEMS (micro electromechanical system) process. The specific binding characteristics of the PSA antigen to its antibody, which is immobilized with Calixcrown self-assembled monolayers (SAMs) on a gold surface deposited on a cantilever, are determined to a high sensitivity. For the bioassay in a liquid environment, a liquid test cell with a 20 microl volume reaction chamber has been fabricated, using a bonding technique between poly(dimethyl siloxane) (PDMS) bilayers. An observed trend of resonant frequency change with respect to time could be explained by the binding kinetics due to the Langmuir isotherm and diffusion and by the effects of a small volume reaction chamber. In the saturated regimes, the resonant frequency of the cantilever increased with increase of the PSA concentration in the reaction chamber, showing that the trend of the resonance frequency change was similar to that of the fluorescence results. The saturated resonance frequency shift of the cantilever was proportional to the PSA antigen concentration of analyte solution.

Antibodies↗

Electrothermal stirring for heterogeneous immunoassays.

A technique is proposed to enhance microfluidic immuno-sensors, for example, immunoassays, in which a ligand immobilized on a microchannel wall specifically binds analyte flowing through the channel. These sensors can be limited in both response time and sensitivity by the diffusion of analyte to the sensing surface. In certain applications, the sensitivity and response of these heterogeneous immunoassays may be improved by using AC electrokinetically-driven microscale fluid motion to enhance antigen motion towards immobilized ligands. Specifically, the electrothermal effect is used to micro-stir analyte near the binding surface. Numerical simulations of antigen in a microchannel flow subjected to the electrothermal effect show that 6 V(rms) applied to electrodes near a binding region can increase binding in the first few minutes by a factor of seven. The effectiveness of electrothermal stirring is a strong function of the Damköhler number. The greatest binding enhancement is possible for high Damköhler numbers, where the reaction is limited by diffusion. Based on these results, the utility of this technique for diffusion-limited microfluidic sensor applications is demonstrated.

Antigen-Antibody Complex↗

Micro flow sensor based on two closely spaced amperometric sensors.

In this communication, a novel micro flow sensor based on two closely spaced amperometric oxygen sensors is proposed and implemented. The simulation results show that the ratio of the responses of these two oxygen sensors is determined by flow rates in the micro-channel. The sensor has been implemented using a micro fabrication technique. The measurement results demonstrate that the technique is able to detect flow rates in the flow range of several microliters per minute when the distance between the working electrodes of two oxygen sensors is 10 microm and the cross-section of the micro-channel is 100 microm x 100 microm. The advantage of the proposed flow sensor is that no additional tracers have to be added or produced during the flow measurement. Information on dissolved oxygen concentration in the liquid is not required either.

Biosensing Techniques↗

Continuous flow in open microfluidics using controlled evaporation.

This paper presents a method for programming the flow rate of liquids inside open microfluidic networks (MFNs). A MFN comprises a number of independent flow paths, each of which starts with an open filling port, has a sealed microchannel in which assays can be performed, and an open capillary pump (CP). The MFN is placed over Peltier elements and its flow paths initially fill owing to capillary forces when liquids are added to the filling ports. A cooling Peltier element underneath the filling ports dynamically prevents evaporation in all filling ports using the ambient temperature and relative humidity as inputs. Another Peltier element underneath the CPs heats the pumps thereby inducing evaporation in the CPs and setting the flow rate in the microchannels. This method achieves flow rates in the microchannels ranging from approximately 1.2 nL s(-1) to approximately 30 pL s(-1), and is able to keep 90% of a 0.6 microL solution placed in an open filling port for 60 min. This simple and efficient method should be applicable to numerous assays or chemical reactions that require small and precise flow of liquids and reagents inside microfluidics.

Capillary Action↗

Micro gas analysis system for measurement of atmospheric hydrogen sulfide and sulfur dioxide.

A honeycomb structure microchannel scrubber was developed to achieve efficient and stable gas collection. A thin porous membrane was pasted on a microchannel by the adhesive force of a fresh polydimethylsiloxane surface. The microchannel scrubber achieved much more efficient gas collection than conventional impingers and diffusion scrubbers. Two sets of the microchannel scrubbers and detectors were integrated in a 10 cm x 9 cm plastic board to create a micro gas analysis system (microGAS) for simultaneous measurements of H2S and SO2. The whole system including a battery was incorporated in a carrying case 34 cm W x 16 cm D x 17 cm H for use in the field. Liquid flows at 30 microl min(-1) were obtained by bimetal micropumps. The estimated detection limits were 0.1 ppbv for H2S and 1 ppbv for SO2. The system was demonstrated for real atmospheric gas analysis, and the results agreed well with data concurrently obtained by ion chromatography coupled with a cylindrical diffusion scrubber. The system we developed allowed automated continuous analyses in the field and achieved a much higher time resolution compared to those by ion chromatographic analysis.

Air Pollutants↗

Determination of Kamlet-Taft solvent parameters pi* of high pressure and supercritical water by the UV-Vis absorption spectral shift of 4-nitroanisole.

Kamlet-Taft solvent parameters, pi*, of high pressure and supercritical water were determined from 16-420 degrees C based on solvatochromic measurements of 4-nitroanisole. For the measurements, an optical cell that could be used at high temperatures and pressures was developed with the specification of minimal dead space. The low dead space cell allowed us to measure the absorption spectra of 4-nitroanisole at high temperature conditions before appreciable decomposition occurred. The behavior of pi* in terms of water density (pi* = 1.77rho- 0.71) was found to be linear, except in the near critical region, in which deviations were observed that could be attributed to local density augmentation. Excess density, which was defined as the difference between local density and bulk density, showed a maximum near the critical density of water. The frequencies of UV-Vis spectra of 4-(dimethylamino)benzonitrile and N,N-dimethyl-4-nitroaniline were correlated with pi* based on a linear solvation energy relationship (LSER) theory. Local density augmentation around 4-nitroanisole and that around 4-(dimethylamino)benzonitrile were similar but the augmentation observed around N,N-dimethyl-4-nitroaniline was larger.

Anisoles↗

Computational-fluid-dynamics (CFD) modelling of an industrial crystallizer: application to the forced-circulation reactor.

The CFD (computational fluid dynamics) technique is used to describe the mixing conditions in a pilot-scale FCC (forced-circulation crystallizer) and to study the impact of flow rate and aspect ratio on local flow conditions and RTD (residence-time distribution) in the crystallizer. The analysis adequately predicts the oscillating flow and two-phase (gas-liquid) interaction at the free surface. A comparison has been made between the CFD predictions and models of RTD. The results support the use of CFD methodology as an aid to optimization of commercial-scale FCC design.

Chemical Fractionation↗

Nanofluidic channels fabrication and manipulation of DNA molecules.

Nanofluidic channel arrays, which have a width of about 40 nm, depth of 60 nm and length of 50 mum, were created using a focused-ion-beam milling instrument on a silicon nitride film swiftly and exactly, as is necessary. Stained -DNA molecules were put inside these sub-100 nm conduits by capillary force and they were stretched and transferred along these conduits, which were dealt with activating reagent Brij aqueous solution in advance. The movements of DNA molecules in these channels were discussed. These nano-structure channels may be useful in the study and analysis of the statics as well as the dynamics of single biomolecules.

DNA↗

Semi-automatic blood lactate assay: experiences from an exercise laboratory.

A semi-automatic system based on flow injection analysis (FIA) for the transportation of small sample aliquots has been combined with fluorometric, enzymatic methods for blood lactate determination and has been described earlier. In the present study duplicate blood samples were obtained from exercising subjects to enable a comparison of lactate concentrations between neutralized and non-neutralized samples. Duplicate samples were also obtained to enable FIA lactate values to be compared to those obtained with a manual enzymatic method, and with a colorimetric method. No significant change was observed if the sample was not neutralized, enabling a more rapid sample turnover. The FIA method was reliable, with a coefficient of variation of 4.9% between duplicate blood samples. FIA lactate values were valid when compared to two other manual assays. FIA has been shown to be a rapid (60 samples x h-1) means of accurately determining blood lactate concentrations with 25 microliter blood samples and is of particular relevance to the exercise laboratory.

Automation↗

Evaluation of the nitrate content in leaf vegetables produced through different agricultural systems.

The nitrate content of leafy vegetables (watercress, lettuce and arugula) produced by different agricultural systems (conventional, organic and hydroponic) was determined. The daily nitrate intake from the consumption of these crop species by the average Brazilian consumer was also estimated. Sampling was carried out between June 2001 to February 2003 in Campinas, São Paulo State, Brazil. Nitrate was extracted from the samples using the procedure recommended by the AOAC. Flow injection analysis with spectrophotometric detection at 460 nm was used for nitrate determination through the ternary complex FeSCNNO+. For lettuce and arugula, the average nitrate content varied (p < 0.05) between the three agricultural systems with the nitrate level in the crops produced by the organic system being lower than in the conventional system that, in turn, was lower than in the hydroponic system. For watercress, no difference (p < 0.05) was found between the organic and hydroponic samples, both having higher nitrate contents (p < 0.05) than conventionally cultivated samples. The nitrate content for each crop species varied among producers, between different parts of the plant and in relation to the season. The estimated daily nitrate intake, calculated from the consumption of the crops produced by the hydroponic system, represented 29% of the acceptable daily intake established for this ion.

Agriculture↗

A simple spectrophotometric method based on pH-indicators for monitoring partial and total alkalinity in anaerobic processes.

Partial alkalinity, as a measure of bicarbonate concentration, and total alkalinity, as a measure of the concentration of bicarbonate and volatile fatty acids, are useful parameters for monitoring anaerobic digestion processes. This paper reports a new method based on pH-indicators and spectrophotometric measurements for the monitoring of partial and total alkalinity. The method was used in an off-line procedure for monitoring of an anaerobic process and displayed less than 5% deviation from the traditional method of measuring partial and total alkalinity, as well as being rapid and reproducible. A flow injection analysis system based on the method was used on-line for monitoring overload in a UASB reactor, which demonstrated changes in alkalinity not easily registered using traditional methods.

Bacteria, Anaerobic↗

In vitro antioxidant activities of antioxidant-enriched toothpastes.

Several forms of periodontal diseases (PD) are often associated with modified phagocytosing leukocytes and contemporary free radical production. Host antioxidant defenses could benefit from toothpastes used as adjuncts to counteract plaque-associated bacteria. The aim of the present study was to determine possible antioxidant activity (AA) of 12 differently antioxidant-enriched toothpastes, regardless of their efficacy as antimicrobial agents. Toothpastes were enriched alternatively with sodium ascorbyl phosphate, alpha-tocopherol acetate, pycnogenol, allantoin and methyl salycilate or a mixture of these. AA was tested in a cell-free system with a ABTS-decolorization assay improved by means of a flow injection analysis device. Comet assay, using NCTC 2544 keratinocytes, was performed to test if it was possible to identify any protection against in vitro DNA fragmentation provoked by a challenge with H(2)O(2) in cultures pre-incubated with toothpaste extracts. Only toothpastes containing sodium ascorbyl phosphate displayed clear AA with I(50) values ranging between 50 and 80 mg of toothpaste/ml water. COMET analysis of cells challenged with H(2)O(2) in presence of toothpaste extracts revealed a limited protection exerted by sodium ascorbyl phosphate. The results described herein indicate that toothpastes containing sodium ascorbyl phosphate possess AA. All the data were obtained in systems in vitro and the demonstration of in vivo AA is desirable. These findings could be useful in the treatment and maintenance of some forms of PD and should be considered when arranging new toothpaste formulations.

Antioxidants↗

Bienzymatic amperometric sensor for protein assay in milk.

A new bienzymatic amperometric sensor is proposed for the assay of the protein content of milk. The sensor is based on two enzymes: carboxypeptidase A and L-amino acid oxidase. The response characteristics obtained for this sensor (detection limit of 1.5 micromol/L, linear concentration range between 1.8 and 2.8 micromol/L), as well as high selectivity over possible interferences from milk, made it applicable as a detector in flow injection analysis (FIA). The response characteristics obtained in the non-equilibrium conditions (FIA system) are: detection limit of 1.5 micromol/L and linear concentration range between 2 and 3.5 micromol/L. Without FIA, the average recovery of proteins from milk and milk products is 99.06 +/- 0.07% and, by utilization of FIA, it increased to 99.73 +/- 0.03. The sensor proved a good reliability for the assay of proteins in milk and milk products.

Amino Acid Oxidoreductases↗

The evaluation of interactions between anaesthetic agent vapours and carrier gases by laser refractometry.

A measurement system and technique have been developed for the evaluation of interactions between anaesthetic agent vapours and carrier gases. The refractivities of mixtures of anaesthetic agent and carrier gas have been compared to values derived from the addition of the absolute refractivities of the individual components. These results show that there is no significant interaction between the anaesthetic agent and the carrier gas at the +/- 0.06% level of uncertainty. Results for the anaesthetic agents isoflurane, sevoflurane, enflurane, halothane, chloroform and desflurane and the carrier gases nitrogen, oxygen and dry air will be presented.

Anesthetics, Inhalation↗