Multi-layer microfluidic glass chips for microanalytical applications

A new, versatile architecture is presented for microfluidic devices made entirely from glass, for use with reagents which would prove highly corrosive for silicon. Chips consist of three layers of glass wafers bonded together by fusion bonding. On the inside wafer faces a network of microfluidic channels is created by photolithography and wet chemical etching. Low dead-volume fluidic connections between the layers are fabricated by spark-assisted etching (SAE), a computer numerical controlled (CNC)-like machining technique new to microfluidic system fabrication. This method is also used to form a vertical, long path-length, optical cuvette through the middle wafer for optical absorbance detection of low-concentration compounds. Advantages of this technique compared with other, more standard, methods are discussed. When the new glass-based device for flow-injection analysis of ammonia was compared with our first-generation chips based on silicon micromachining, concentration sensitivity was higher, because of the longer path-length of the optical cuvette. The dependence of dispersion on velocity profile and on channel cross-sectional geometry is discussed. The rapid implementation of the devices for an organic synthesis reaction, the Wittig reaction, is also briefly described.     

Overview of calorimeter chips for various applications

This paper gives an overview of some of the recent developments in the area of chip calorimetry. Using several chip calorimeters developed at Xensor Integration and tested by users, examples of chip calorimetry and its applications will be given. Examples of chip calorimeters developed at various universities are given to widen the overview. The examples will be used to give more insight in the design and fabrication of various chip calorimeters.     

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(?) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.     

Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates

A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.     

An electrochemical microsensor for chloride

The potential-response of a microdisk electrode made with a chloride-doped polypyrrole (PPY) film on a carbon fibre (CF) has been examined. The effect of the polymerization conditions on the response characteristics is discussed. The optimum conditions for preparing the electrode are: cycling potential from +0.8 to +1.0 V in 0.1-0.2M pyrrole (Py) containing 0.1M LiCl, electropolymerization time 15-20 min. The electrode gives a Nernstian response of 56-58 mV/pCl and a detection limit of 3.6 x 10(-5)M chloride. It has the advantages of low resistance, short conditioning time and fast response. It has been used satisfactorily for detection of chloride in serum.     

Gene expression analysis for high throughput screening applications

To meet growing needs for high throughput gene expression profiling, we established a new automated high throughput TaqMan RT-PCR method for quantitative mRNA expression analysis. In this method, the Allegro( trade mark ) (Zymark) system conducts all sample tracking and liquid handling steps, and ABI PRISM 7900 HT (Applied Biosystems) is used to conduct real-time determination of the C(t) value when amplification of PCR products is first detected and accumulation of inhibitory PCR products is unlikely to occur. The ABI PRISM 7900 HT Sequence Detection System features a real-time PCR instrument with 384-well-plate compatibility and robotic loading, and continuous wavelength detection, which enables the use of multiple fluorophores in a single reaction. The Allegro System offers an assembly line approach with a modular design that allows reconfiguration of the components to accommodate variations in the assay flow. In the present study, we have established and validated a new automated High Throughput (HT) TaqMan RT-PCR- based method for quantitative mRNA expression analysis. The data demonstrate that HT-Taqman PCR is a powerful tool that can be used for measuring low concentrations of mRNA, and is highly accurate, reproducible, and amenable to high throughput analysis. Results suggest that HT-TaqMan is a reliable method for the quantification of low-expression genes and a powerful tool with HT capability for target identification/validation, structure-activity relationship (SAR) study, compound selection for efficacy studies, and biomarker identification in drug discovery and development.     

Planar chromatography for drug residue screening in food applications

Summary Planar chromatography (PC) is of interest for veterinary drug screening. An example is given of the use of a PC screening method followed by a high performance liquid chromatographic (HPLC) method for confirmation of positives. The results for 300 samples were obtained in 14 days whereas the HPLC analysis alone would have required 50 days work. The future of PC in veterinary drug residue analysis as a screening method and also as confirmatory (quantitative) method is discussed.     

Bifunctional atomic force microscopy probes for molecular screening applications

Force mapping with the atomic force microscope (AFM) allows the simultaneous acquisition of topography and probe-sample interaction data. For example, AFM probes functionalised with an antigen can be employed to map the spatial distribution of recognition events on a substrate functionalised with its specific antibody. However, to date this method has been limited to the detection of single receptor-ligand species. Were the detection of multiple receptor-ligand interactions possible, force mapping would offer great scope as a sensitive tool for bioassay and screening applications. We have developed an immobilisation strategy, which allows two different molecular species (in this case human serum albumin and the β subunit of human chorionic gonadotropin) to be present simultaneously on an AFM probe. Single point force spectroscopy results have revealed the ability of such probes to discriminate between their corresponding recognition points (anti-HSA and anti-βhCG IgG antibodies). As a control, force measurements were re-recorded in the presence of the known antigen (free in solution) for each antibody species and a marked decrease in the frequency of specific interaction is observed. As an additional control interactions between anti-βhCG IgG and the multifunctional probe are taken in the presence of free βhCG (“true” antigen) and free HSA (“false” antigen). It is shown that measurements recorded in the presence of a non-related protein species results in no change in either the force observed or the frequency of specific interactions, further confirmation that the specificity of force observed is due to the separation of antibody-antigen complex.     

Enzyme-dispersed carbon-nanotube electrodes: a needle microsensor for monitoring glucose

The preparation of an enzyme-dispersed carbon-nanotube (CNT) electrode, based on mixing glucose oxidase (GOx) within CNT, is described. The new binderless biocomposite was packed within a 21-gauge needle and used for amperometric monitoring of glucose. The resulting microsensor offers a low-potential highly selective and sensitive detection of glucose. The high sensitivity and selectivity are coupled to a wide linear range, prolonged lifetime and oxygen independence. About 80% of the GOx activity is retained during a 24 h thermal stress at 90 degrees C, reflecting the enzyme-stabilization action of CNT. The marked electrocatalytic action towards hydrogen peroxide allows highly selective detection of the glucose substrate at -0.1 V (vs. Ag/AgCl) with no interferences from coexisting ascorbic acid, acetaminophen or uric acid. Linearity prevails up to 40 mM glucose (with analytically useful signals observed up to 0.1 M). Factors affecting the performance of the CNT-based glucose biosensor were assessed and optimized. The attractive performance of the new needle electrode offers great promise for continuous monitoring of glucose in connection to the management of diabetes, and for the biosensing of other metabolites.     

Enzyme microsensor for glucose with an electrochemically synthesized enzyme-polyaniline film

An enzyme microsensor for glucose was fabricated by the electrochemical polymerization method. A glucose oxidase-entrapped polyaniline (GOD-polyaniline) film was deposited on the top of a platinum fibre (50 μm in diameter) by the electrochemical oxidative polymerization of aniline in a pH 7 buffer solution in the presence of glucose oxidase. The GOD-polyaniline films retained GOD activity and oxygen permeability but prevented large molecules from permeating. Glucose was auperometrically determined with the electrochemically fabricated microsensor in the concentration range 10⁻⁴ to 5 × 10⁻³ M.     

Fiber-optic microsensor for high resolution pCO2 sensing in marine environment

A fast responding fiber-optic microsensor for sensing pCO₂ in marine sediments with high spatial resolution is presented. The tip diameter varies typically between 20 and 50 μm. In order to make the pH-indicator ¶8-hydroxypyrene-1,3,6-trisulfonate soluble in the ethyl cellulose matrix, it was lipophilized with tetraoctylammonium as the counterion [HPTS-(TOA)₄]. The microsensor was tuned to sense very low levels of dissolved carbon dioxide which are typically present in marine systems. The detection limit is 0.04 hPa pCO₂ which corresponds to 60 ppb CO₂ of dissolved carbon dioxide. A soluble Teflon derivative with an extraordinarily high gas permeability was chosen as a protective coating to eliminate interferences by ionic species like chloride or pH. Response times of less than 1 min were observed. The performance of the new microsensor is described with respect to reproducibility of the calibration curves, dynamic range, temperature behavior, long term stability and storage stability. The effect of hydrogen sulfide as an interferent, which is frequently present in anaerobic sediment layers, was studied in detail.     

Fiber-optic microsensor for high resolution pCO2 sensing in marine environment

A fast responding fiber-optic microsensor for sensing pCO2 in marine sediments with high spatial resolution is presented. The tip diameter varies typically between 20 and 50 microm. In order to make the pH-indicator 8-hydroxypyrene-1,3,6-trisulfonate soluble in the ethyl cellulose matrix, it was lipophilized with tetraoctylammonium as the counterion [HPTS-(TOA)4]. The microsensor was tuned to sense very low levels of dissolved carbon dioxide which are typically present in marine systems. The detection limit is 0.04 hPa pCO2 which corresponds to 60 ppb CO2 of dissolved carbon dioxide. A soluble Teflon derivative with an extraordinarily high gas permeability was chosen as a protective coating to eliminate interferences by ionic species like chloride or pH. Response times of less than 1 min were observed. The performance of the new microsensor is described with respect to reproducibility of the calibration curves, dynamic range, temperature behavior, long term stability and storage stability. The effect of hydrogen sulfide as an interferent, which is frequently present in anaerobic sediment layers, was studied in detail.     

Remote optical fibre microsensor for monitoring BTEX in confined industrial atmospheres

A portable optical fibre sensor has been developed for remote monitoring of benzene, toluene, ethylbenzene, p-xylene, m-xylene and o-xylene (BTEX). Firstly, the analyser was tested for calibration and its analytical performance for BTEX monitoring compared with a more classical analytical method, namely gas chromatography coupled to a flame ionization detector (GC-FID). The developed remote sensor shows several analytical advantages such as, high analytical sensitivity and accuracy, good linearity and stability of the analytical signal and short analytical time. Secondly, the optical fibre based sensor was applied to air monitoring for detection and quantification of BTEX in a confined industrial environment. The analytical signal measurement was performed by wireless at 20 m of distance from the local of analysis. Besides, the reported sensor showed a high degree of portability, compact design and high analytical performance for remote BTEX monitoring, in situ and in real-time.     

An Enzyme microsensor for urea based on an ammonia gas electrode

A urea microsensor was fabricated by immobilizing urease at the tip (10-μm diameter) of a rapidly responding ammonia gas microelectrode based on antimony. The construction and evaluation of both the urea senson and the ammonia electrode are described in detail. The urea sensor responds to 10^?2?10^?4 M urea in 30–45 s.     

A microsensor for urea based on an ion-selective field effect transistor

The pH response of a pH-sensitive field-effect transistor (FET) is not affected by a ca. 1-μm thick membrane formed from λ-aminopropyltriethoxysilane and glutaraldehyde over the gate insular by a vapor deposition method. The response between pH 5.5 and 8.5 is ca. 61 mV pH^?1 at 37°C in 5 mM Tris-HCl buffer. When urease is immobilized on the membrane, the sensor gives a linear relationship between the initial rate of the output gate voltage change and the logarithmic value of urea concentration between 16.7 and 167 mM. Determination of urea is possible within 30 s. The optimum pH is 6.0–6.5, at 37°C. The system can be used for 20 days with only slight loss of enzymatic activity.     

High-throughput computational screening of oxide double perovskites for optoelectronic and photocatalysis applications

Oxide double perovskites A2B'BO6 are a class of emerging materials in the fields of optoelectronics and catalysis.Due to the chemical flexibilities of perovski...     

Oxygen-independent glucose microsensor based on glucose oxidase

A glucose oxidase-based needle-type microsensor is described that is independent of the local oxygen concentration. The sensor consists of an enzyme-coated platinum microelectrode that is inserted in a glass capillary with a tip of 5–25 μm, and connected to the environment via an agar membrane. Oxygen is supplied to the enzyme coating from the shaft of the capillary. Depending on the electrode configuration, the sensor has a response time of 1–10 s, and the measuring range extends from 0.01 to 0.4 mmol l^?1 or from 0.4 to 10 mmol l^?1. The signal is not affected by stirring.     

A microsensor for adenosine-5′-triphosphate pH-sensitive field effect transistors

The sensor for adenosine-5′-triphosphate (ATP) is based on H⁺-ATPase immobilized via a polyvinylbutyral resin on a pH-sensitive field effect transistor. A linear relationship was obtained between the initial rate of change of the differential gate output voltage and the logarithm of the ATP concentration over the range 0.2–1.0 mM ATP. The optimum pH was 9.0 at 40°C but pH 7.0 was preferred for routine measurements. Only slight responses were obtained for 1 mM glucose, creatinine or urea. The ATP-sensing system exhibited a response to 1 mM ATP for at least 18 days.