Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay.

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of ß-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.

Chemical amplification: continuous-flow PCR on a chip.

A micromachined chemical amplifier was successfully used to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device is analogous to an electronic amplifier and relies on the movement of sample through thermostated temperature zones on a glass microchip. Input and output of material (DNA) is continuous, and amplification is independent of input concentration. A 20-cycle PCR amplification of a 176-base pair fragment from the DNA gyrase gene of Neisseria gonorrhoeae was performed at various flow rates, resulting in total reaction times of 90 seconds to 18.7 minutes.

Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip.

Micromachining technology was used to prepare chemical analysis systems on glass chips (1 centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and electrophoretic separation to distinguish sample components. Capillaries 1 to 10 centimeters long etched in the glass (cross section, 10 micrometers by 30 micrometers) allow for capillary electrophoresis-based separations of amino acids with up to 75,000 theoretical plates in about 15 seconds, and separations of about 600 plates can be effected within 4 seconds. Sample treatment steps within a manifold of intersecting capillaries were demonstrated for a simple sample dilution process. Manipulation of the applied voltages controlled the directions of fluid flow within the manifold. The principles demonstrated in this study can be used to develop a miniaturized system for sample handling and separation with no moving parts.

Bilayer microfluidic chip for diffusion-controlled activation of yeast species.

A bilayer microfluidic chip is used, in which multiple laminar streams are generated to define local microenvironments. The bilayer architecture of the microchip separates cell handling and positioning from cell activation by soluble chemicals. Cell activation is diffusion controlled through a porous membrane. By employing time-lapse fluorescence microscopy, gene expression of the enhanced green fluorescent protein (eGFP) in Saccharomyces cerevisiae is studied under various conditions. We demonstrate that the yeast cells remain viable in the microchip for at least 17 h, and that gene expression can be initiated by the supply of the inducer galactose at a spatial precision of a few micrometers.

Developments in technology and applications of microsystems.

In the past year, microchips as applied to miniaturised total analysis systems, or microTAS, have benefited from technological improvements in their fabrication and been applied to analysis in many different biological areas. From a technological perspective, salient work includes fast, cheap and easy micromachining in polymers and integrated optical detection. From the bioapplications perspective, advances in DNA and protein separations, cell manipulations, immunoassays and polymerase chain reaction using on-chip electrophoretic separation stand out.

Direct coupling of a free-flow isotachophoresis (FFITP) device with electrospray ionization mass spectrometry (ESI-MS).

We present the online coupling of a free-flow isotachophoresis (FFITP) device to an electrospray ionization mass spectrometer (ESI-MS) for continuous analysis without extensive sample preparation. Free-flow-electrophoresis techniques are used for continuous electrophoretic separations using an electric field applied perpendicular to the buffer and sample flow, with FFITP using a discontinuous electrolyte system to concurrently focus a target analyte and remove interferences. The online coupling of FFITP to ESI-MS decouples the separation and detection timeframe because the electrophoretic separation takes place perpendicular to the flow direction, which can be beneficial for monitoring (bio)chemical changes and/or extensive MS(n) studies. We demonstrated the coupling of FFITP with ESI-MS for simultaneous concentration of target analytes and sample clean-up. Furthermore, we show hydrodynamic control of the fluidic fraction injected into the MS, allowing for fluidically controlled scanning of the ITP window. Future applications of this approach are expected in monitoring biochemical changes and proteomics.

Modular approach to fabrication of three-dimensional microchannel systems in PDMS-application to sheath flow microchips.

A modular approach to fabrication of three-dimensional microchannel systems in polydimethylsiloxane (PDMS) is presented. It is based on building blocks with microstructuring on up to three faces. The assembled 3D-microchip consists of three building blocks in two layers. For assembly of the bottom layer two building blocks are joined horizontally, whereby the side structuring of the first is sealed against the flat side surface of the other. This results in the formation of a vertical interconnection opening between the building blocks to supplement the microstructuring on the lower faces. The 3D microchannel system is completed by placing a third building block, with microstructuring only on its lower face, on top of the assembled layer. While plasma assisted bonding is used between the two building blocks of the bottom layer, inherent adhesion is sufficient between the layers and for attaching the assembled 3D-microchip to a substrate. This modular approach was applied to the fabrication of a 3D-sheath flow microchip. It comprises a 20 microm deep microchannel system with sample inlet, open sensing area and outlet in the bottom layer and sheath flow inlet in the top layer. 100 microM fluorescein at 6 microL min(-1) was used as sample flow and water at increasing flow rates as sheath flow. With ratios of sheath to sample flow up to 20:1 sample layers down to 1 microm thickness could be generated. Sample layer thickness was determined via volume detection on an epi-fluorescence microscope followed by image analysis.

Wavelet transform for Shah convolution velocity measurements of single particles and solutes in a microfluidic chip.

Wavelet transform analysis is applied to determine the speed of fluorescent polystyrene microspheres and fluorescent solutes in a microchip. The data analysed consist of the periodical signal (Shah convolution) obtained when fluorescent particles or solute plugs move in a channel that is covered with a chromium grid pattern. This setup converts velocity into a (fluorescence emission) frequency, and previous analyses therefore used Fourier transform to extract the frequency information. In this paper it is shown that wavelet transform has some advantages over Fourier transform. With wavelet transform, time information can be obtained in addition to frequency information. Thus the speed of individual particles was determined together with their moments of appearance and disappearance in the system. With solutes small changes of velocity during the analysis were detected, and an improvement in peak frequency resolution was obtained.

Miniaturised nucleic acid analysis.

The application of micro total analysis systems has grown exponentially over the past few years, particularly diversifying in disciplines related to bioassays. The primary focus of this review is to detail recent new approaches to sample preparation, nucleic acid amplification and detection within microfluidic devices or at the microscale level. We also introduce some applications that have as yet to be explored in a miniaturised environment, but should benefit from improvements in analytical efficiency and functionality when transferred to planar-chip formats. The studies described in this review were published in commonly available journals as well as in the proceedings of three major conferences relevant to microfluidics (Micro Total Analysis Systems, Transducers and The Nanotechnology Conference and Trade Show). Although an emphasis has been placed on papers published since 2002, pertinent articles preceding this publication year have also been included.

Electrophoretic manipulation of single DNA molecules in nanofabricated capillaries.

We demonstrate the use of nanofabricated capillaries, integrated as part of a microfluidic structure, to study the electrophoretic behaviour of single, fluorescently-labelled, molecules of DNA as a function of capillary size. The nanocapillaries, fabricated using a focused ion beam, have cross-sections down to 150 x 180 nm. Control of single-molecule direction and velocity was achieved using voltage manipulation. DNA mobility was found to increase with decreasing cross-section, which we interpret in terms of reduced electro-osmotic counter-flow. Such nanofabricated capillaries as part of larger fluidic structures have great potential for biotechnology, particularly single molecule manipulation and analysis.

An AC electroosmotic micropump for circular chromatographic applications.

Flow rates of up to 50 microm s(-1) have been successfully achieved in a closed-loop channel using an AC electroosmotic pump. The AC electroosmotic pump is made of an interdigitated array of unequal width electrodes located at the bottom of a channel, with an AC voltage applied between the small and the large electrodes. The flow rate was found to increase linearly with the applied voltage and to decrease linearly with the applied frequency. The pump is expected to be suitable for circular chromatography for the following reasons: the driving forces are distributed over the channel length and the pumping direction is set by the direction of the interdigitated electrodes. Pumping in a closed-loop channel can be achieved by arranging the electrode pattern in a circle. In addition the inherent working principle of AC electroosmotic pumping enables the independent optimisation of the channel height or the flow velocity.

Poly(dimethylsiloxane) electrospray devices fabricated with diamond-like carbon-poly(dimethylsiloxane) coated SU-8 masters.

This study presents coupling of a poly(dimethylsiloxane) (PDMS) micro-chip with electrospray ionization-mass spectrometry (ESI-MS). Stable electrospray is generated directly from a PDMS micro-channel without pressure assistance. Hydrophobic PDMS aids the formation of a small Taylor cone in the ESI process and facilitates straightforward and low-cost batch production of the ESI-MS chips. PDMS chips were replicated with masters fabricated from SU-8 negative photoresist. A novel coating, an amorphous diamond-like carbon-poly(dimethylsiloxane) hybrid, deposited on the masters by the filtered pulsed plasma arc discharge technique, improved significantly the lifetime of the masters in PDMS replications. PDMS chip fabrication conditions were observed to affect the amount of background peaks in the MS spectra. With an optimized fabrication process (PDMS curing agent/silicone elastomer base ratio of 1/8 (w/w), curing at 70 degree C for 48 h) low background spectra were recorded for the analytes. The performance of PDMS devices was examined in the ESI-MS analysis of some pharmaceutical compounds and amino acids.

Integrated potentiometric detector for use in chip-based flow cells

A new kind of potentiometric chip sensor for ion-selective electrodes (ISE) based on a solvent polymeric membrane is described. The chip sensor is designed to trap the organic cocktail inside the chip and to permit sample solution to flow past the membrane. The design allows the sensor to overcome technical problems of ruggedness and would therefore be ideal for industrial processes. The sensor performance for a Ba2+-ISE membrane based on a Vogtle ionophore showed electrochemical behavior similar to that observed in conventional electrodes and microelectrode arrangements.

A dc microplasma on a chip employed as an optical emission detector for gas chromatography

A micromachined plasma chip is coupled to a conventional gas chromatograph to investigate its performance as an optical emission detector. The device employs a 180-nL plasma chamber in which an atmospheric pressure dc glow discharge is generated in helium. Applied power is 9 mW (770 V, 12 microA) and helium flow rate 320 nL s(-1). A number of carbon-containing compounds are detected in the column effluent by recording the emission at 519 nm. For hexane, the detector has a linear dynamic range of over two decades and a minimum detectability of 10(-12) g s(-1) (800 ppb). The detector signal shows a marked peak broadening and tailing when compared with the signal of a flame ionization detector. This is mainly attributed to dead volumes and chromatographic processes introduced by the connecting tubing and the chip glass channels. The device was operated for more than 24 h without a significant change in performance. Operation is stable and instrumental requirements are simple. Future use of the detector chip in conventional gas chromatography or as an integrated detector in on-chip gas chromatography is discussed.

Immune cell functions in industrial workers after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: dissociation of antigen-specific T-cell responses in cultures of diluted whole blood and of isolated peripheral blood mononuclear cells.

A comparative analysis was performed of the phenotype and function of peripheral blood leukocytes of two age-matched cohorts of industrial workers in chemical plants, one of which was exposed occupationally to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Median actual TCDD burdens were 116 ng/kg and 4 ng/kg, respectively. The phenotype analysis of peripheral blood mononuclear cells (PBMC) revealed no significant differences in the proportions of CD3, CD4, or CD8+ T lymphocytes, of CD16+ natural killer cells, and of CD19+ B lymphocytes. However, in PBMC of the TCDD-exposed workers; the proportion of CD8+ memory T cells (CD45R0+) was significantly higher, and that of lymphocytes with naive phenotype (CD45RA+) was significantly lower than in PBMC of the control group. Polyclonal and antigen-specific T-cell activation was assessed in parallel in isolated PBMC as well as in diluted whole blood cultures. In both culture systems the polyclonally stimulated cytokine release did not differ significantly between the two cohorts; however, we found a significantly reduced interferon gamma release in diluted whole blood cultures but not in isolated PBMC cultures of the TCDD-exposed cohort when we performed an antigen-specific T-cell stimulation with tetanus-toxoid. Therefore, we propose that exposure of individuals to high doses of TCDD can partially impair in the "blood milieu" those T-cell/monocyte interactions that are essential for antigen-specific T-cell responses, whereas isolated PBMC of the same donors appear functionally less affected.

Immunologic findings in workers formerly exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and its congeners.

One hundred ninety-two workers in a German pesticide factory who were exposed to polychlorinated dibenzodioxins and -furans (PCDD/PCDF) were investigated for former and present diseases and laboratory changes of the immune system. Moreover, in a subgroup of 29 highly exposed and 28 control persons, proliferation studies were performed. In addition to assays such as blood count, immunoglobulins, serum electrophoresis, monoclonal bands, surface markers, autoantibodies, and lymphocyte proliferation, two new methods, the rise of tetanus antibody concentration after vaccination and the in vitro resistance of lymphocytes to chromate, were used to diagnose the morphologic and functional state of the immune system. There was no stringent correlation of actual PCDD/PCDF concentrations with the occurrence of infections or with one of the immune parameters. In addition, outcomes of the tetanus vaccination and the chromate resistance test were not correlated with PCDD/PCDF. However, the chromate resistance of lymphocytes stimulated by phytohemagglutinin of highly exposed persons was significantly lower than that for the control group. These findings indicate that the function of lymphocytes can be stressed and possibly impaired by high exposure to PCDD/PCDF.

An integrated silicon thermophile as biosensor for the thermal monitoring of glucose, urea and penicillin.

A new kind of calorimetric biosensor for the measurement of the heat (molar enthalpy change) of enzymatic reactions is presented. The device operates according to the Seebeck effect, the same principle on which thermocouples are based. The thermopile used in this work consists of an array of p-type silicon/aluminium strips integrated on a thin silicon membrane (5 microns). Its sensitivity is about 1 V output voltage per watt of heating power, corresponding to a temperature resolution in the order of 10(-5) K and a heating power resolution of some tenths of a mu W in the flow system used. Furthermore, this performance is obtained without any control of external temperature because of the high common-mode thermal noise rejection ratio of the thermopile. The universal technique of calorimetry combined with the specificity of biochemical reactions makes this biosensor very versatile, with a broad range of possible applications. Glucose oxidase together with catalase for the determination of glucose, urease and penicillinase for the monitoring of urea and penicillin G, respectively, were immobilized directly onto the back side of the thermopile. The sensor was operated in conjunction with flow injection analysis which, in addition to its traditional advantages, allows preconditioning of the samples. Thus, artefacts due to mixing effects were suppressed and interference caused by differences in ionic strength between sample and carrier was strongly decreased. Detection limits between 1 and 2 mM were reported in the flow injection conditions described.

Flow injection analysis and in-line biosensors for bioprocess control: a comparison.

Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems.

Shah convolution differentiation Fourier transform for rear analysis in microchip capillary electrophoresis.

This paper first reports the application of Shah convolution differentiation Fourier transform for rear analysis. Rear analysis eliminates the need to create a well-defined and reproducible sample plug, thus making the operation simpler. The number of solution reservoirs, for microchip capillary electrophoresis (CE), could be reduced from the usual four to three. Sample bias in CE could be avoided too. The separation channel was first filled with the fluorescent sample solution, and subsequently flushed out with the buffer. The rear of each analyte zone gives rise to its flight of sigmoid-shaped steps in the time-domain. The time-domain detector signal was first differentiated and then Fourier transform was performed. The Fourier transform results were represented in the form of a magnitude plot. It is proposed that this would be as equally applicable to other separation techniques (e.g., chromatography) and detection methods (e.g., absorption).

Novel instrumentation for real-time monitoring using miniaturized flow systems with integrated biosensors.

A prototype miniaturized Total Chemical Analysis System (muTAS) has been developed and applied to on-line monitoring of glucose and lactate in the core blood of anaesthetized dogs. The system consists of a highly efficient microdialysis sampling interface sited in a small-scale extracorporeal shunt circuit ('MiniShunt'), a silicon machined microflow manifold and integrated biosensor array for glucose and lactate detection with associated computer software for analytical process control. During in-vivo testing the device allowed real-time on-screen monitoring of glucose and lactate with system response times of less than 5 min, made possible by the small dead volume of the microflow system. On-line glucose and lactate measurements were made in the basal state as well as during intravenous infusion of glucose or lactate. The prototype muTAS is currently suitable for trend monitoring but refinements are necessary before application of the system for determination of individual lactate values.