Proceedings from the 1st International Meeting on Microsensors and Microsystems. Tainan, Taiwan, 13-14 January 2003.
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Biosensing Techniques↗
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Biosensing and monitoring of cell populations using the hydrogel bacterial microchip.
Advanced development of the hydrogel bacterial microchip (HBMChip) technique is proposed. The microchip represents an array of hemispherical gel elements 0.3-60 nl in volume attached to hydrophobic glass surface and containing live immobilized microbial cells. Separate gel elements contain each up to 10(5) cells and retain them inside even while the cells are dividing. Porous structure of the gel provides easy access of nutrients and tested substances to the immobilized cells. Optical signals from the cells are easily measurable and allow monitoring of intracellular metabolism using vital fluorescent stains or engineered constructs encoding bioluminescent or fluorescent reporters. Two possible application modes of the HBMChip have been investigated, i.e. the observation of bacteria and biosensing. The dynamics of nucleic acids synthesis in growing E. coli cells has been analyzed using vital fluorescent stain SYTO 9. A special function has been suggested for evaluation of the cell growth parameters. Biosensing properties of the HBMChip have been illustrated by quantitative analysis of antibiotics and the detection of sodium meta-arsenite.
Anti-Bacterial Agents↗
Electrochemical microdevice with separable electrode and antibody chips for simultaneous detection of pepsinogens 1 and 2.
An electrochemical microdevice with separable electrode and antibody chips has been developed and applied to detect atrophic gastritis-related proteins, pepsinogen 1 (PG1) and pepsinogen 2 (PG2), based on sandwich-type enzyme-linked immunosorbent assays (ELISAs) with horseradish peroxidase (HRP)-labeled antibody. To fabricate the electrochemical device for simultaneous analysis of several proteins, the electrode chip with eight electrode elements was assembled along with an antibody chip with eight cavities containing immobilized anti-PG1 or anti-PG2. The immunoreactions occurring in the cavities of the device were detected simultaneously by amperometry. The labeled HRP in the cavity in the presence of hydrogen peroxide catalyzed the oxidation of ferrocenemethanol (FMA) to FMA+, which was detected electrochemically by the electrode chip. The amperometric responses of respective cavities in the device increased with increasing concentration of PG1 or PG2 of 0-50 ng/ml, ensuring the simultaneous detection of PG1 and PG2. The detection limits for both PG1 and PG2 were 0.6 ng/ml (S/N=2). The electrode chip was recovered easily by disassembling the electrochemical device; thereby, it was used repeatedly, whereas the antibody chip was discarded. No marked decrease in electrochemical responses was detected after repeated use. Reuse of the electrode chip is beneficial to reduce costs of protein analysis.
Antibodies↗
Enzymatic microreactors in chemical analysis and kinetic studies.
The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.
Bioreactors↗
Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.
The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.
Chemical Engineering↗
Characterization of the B2 receptor and activity of bradykinin analogs in SHP-77 cell line by Cytosensor microphysiometer.
The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of measuring the rate at which cells acidify their environment in response to ligand-receptor binding. By measuring the extracellular acidification response (ECAR) we characterized some aspects of ligand-B2 receptor interaction in SHP-77 cell line. SHP-77 cells maximally acidified their environment within 30 s after the exposure to bradykinin (BK) or the BK agonist, B9972, with the maximum effect seen at a ligands concentration of 1 microM. Fetal bovine serum (FBS) modulated the binding of BK or B9972, showing that B9972 is a partial agonist. In addition, the binding of BK agonist or antagonist to the B2 receptor showed different ECAR and different interaction with other intracellular and plasma membrane proteins. Our microphysiometrical results showed that two parameters, antagonist binding affinity (pD2) and antagonist potency (pIC50) are required to characterize BK antagonist activity for the B2 receptor in the SHP-77 cell line. The previously used parameter of B2 antagonist activity, pA2, had high variation and poor correlation with the inhibition of SHP-77 cell growth in vitro and suppression of tumor growth when SHP-77 cells were injected to mice. Our results permit us to conclude that BK agonists and antagonists differ in their interactions with the B2 receptor and consequently elicit different cell responses. Based on our results, we have developed a new microphysiometrical assay for analyzing the activity of BK agonists and antagonist in SHP-77 cells, which may facilitate the discovery of new potent anticancer drugs.
Amino Acid Sequence↗
Protein components for nanodevices.
A long-term goal of nanobiotechnology is to build tiny devices that respond to the environment, perform computations and carry out tasks. Considerable progress has been made in building protein components for such devices, and here we describe examples, including self-assembling protein arrays, pores with triggers and switches, and motor proteins harnessed for specific tasks. A major issue that has been successfully addressed in this recent work is the interface between the proteins and other components of the system, such as a metal surface. While further progress is expected in the coming years, the assembly of devices from the components has seen more limited accomplishments. For example, although a wide variety of sensors based on nanobiotechnology has been developed, unresolved problems still confront the construction of complex nanobioelectronic circuits, and the development of nanorobotics with biological components remains a distant dream.
Microchip Analytical Procedures↗
Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence detection through comigration with a digest.
We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays.
DNA↗
Column switching in zone electrophoresis on a chip.
This feasibility study deals with column switching in zone electrophoresis (ZE) separations on a column coupling (CC) chip. The column switching implemented into the ZE separations an on-chip sample clean up applicable for both the multicomponent and high salinity samples. In addition, complemented by different separation mechanisms in the coupled columns (channels), it provided benefits of two-dimensional separations. Properly timed column switching gave column-to-column transfers of the analytes, characterized by 99-102% recoveries, delivered to the second separation stage on the chip the analyte containing fractions contaminated only with minimum amounts of the matrix constituents. A diffusion driven transport of the matrix constituents to the second channel of the chip (due to direct contacts of the electrolyte solutions in the bifurcation region), representing 0.1-0.2% of the loaded sample constituents, was found to accompany the sample clean up performed on the CC chip. This source of potential disturbances to the separation in the second channel, however, is not detectable in a majority of practical situations. With respect to a 900 nl volume of the sample channel on the CC chip, the electric field and isotachophoresis (ITP) stackings were employed to minimize the injection dispersion in the separations and concentrate the analytes. Here, the column switching, removing a major part of the stacker from the separation system, provided a tool effective in a control of the destacking of analytes. Highly reproducible ZE separations as attained in this work also for the chip-to-chip and equipment-to-equipment frames can be ascribed, at least in part, to suppressions of electroosmotic and hydrodynamic flows of the solutions in which the separations were performed.
Electrophoresis↗
Microchannel-electrode alignment and separation parameters comparison in microchip capillary electrophoresis by scanning electrochemical microscopy.
The end of separation channel in a microchip was electrochemically mapped using the feedback imaging mode of scanning electrochemical microscopy (SECM). This method provides a convenient way for microchannel-electrode alignment in microchip capillary electrophoresis. Influence of electrode-to-channel positions on separation parameters in this capillary electrophoresis-electrochemical detection (CE-ED) was then investigated. For the trapezoid shaped microchannel, detection in the central area resulted in the best apparent separation efficiency and peak shape. In the electrode-to-channel distance ranging from 65 to 15mum, the limiting peak currents of dopamine increased with the decrease of the detection distance due to the limited diffusion and convection of the sample band. Results showed that radial position and axial distance of the detection electrode to microchannel was important for the improvement of separation parameters in CE amperometric detection.
Automation↗
Analysis of human hippocampus gangliosides by fully-automated chip-based nanoelectrospray tandem mass spectrometry.
Modern microfluidic devices are currently introduced in electrospray (ESI) mass spectrometry (MS), tending to substitute the classical capillary-based ESI infusion. Automated systems using the combination of robotized sample handling and chip-based ESI are significantly increasing the analysis reproducibility, precision, throughput, and efficiency. In the last couple of years our group developed the chip-based ESI-MS approach for glycomics in biomedical research and applied it for oligosaccharide, glycopeptide and ganglioside investigation. Here we report upon the optimization and application of this modern technique for the analysis of differential ganglioside expression patterns in human fetal and adult hippocampus. By this methodology, ganglioside species exhibiting high degree of heterogeneity in the ceramide motifs and biologically-relevant modifications could be identified in human hippocampus. The ultra-high reproducibility of the experiments uniquely provided by the chip-ESI approach allowed for a reliable MS-based ganglioside comparative assay. Moreover, the particular feature of chip ESI-tandem MS to provide structural information at high sensitivity was useful for detailed characterization of hippocampus-associated species. The experimental data presented in this study indicate the benefits of microfluidic/MS for determination of the topospecific brain ganglioside composition and development-related changes in their expression, which might be of high value in clinical investigation and for studies related to ganglioside-based therapy of central nervous system diseases.
Adult↗
Lab-on-a-chip technology for determination of protein isoform profiles.
A novel lab-on-a-chip technique for rapid (<15 min) and quantitative isoform-profile determination is presented. Ion-exchange chromatographic separation of protein-isoforms and a sensitive immunoassay detection are combined in a porous monolith chip. Thin lines of immobilized antibodies are used for specific capturing of target molecules, which can be detected by the reaction with antibodies bound to carbon black nano-strings. The bound carbon black is quantified by the use of an image scanner. As demonstrated with transferrin isoforms, differing only by 0.1 pH unit in their pI, this technology can distinguish minor differences in protein carbohydrate structure and enable specific determination of proteins in a complex environment, requiring only a few picogram of isoform for detection.
Chromatography, Ion Exchange↗
Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine.
The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.
Animals↗
Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum.
The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.
Adult↗
Automated nanospray using chip-based emitters for the quantitative analysis of pharmaceutical compounds.
An automated nanospray system based on chip technology (the NanoMate) was successfully interfaced to a modified Particle Discriminator Interface on a triple quadrupole mass spectrometer. A number of the interface parameters were optimized to improve the sampling efficiency for ions from the chip-based system. Analytical performance was assessed using a number of biochemicals as well as via a methodology for a pharmaceutical that passed validation as required by Good Laboratory Practices. Infusion analyses in flow rates <1 microL/min provided advantages in terms of throughput and sample consumption when compared to other methodologies based on liquid chromatography.
Deoxycytidine↗
Continuous beds (monoliths): stationary phases for liquid chromatography formed using the hydrophobic interaction-based phase separation mechanism.
The pioneering research work published by Hjertén et al. [J. Chromatogr. 473 (1989) 273] in 1989 dealing with development and application of the continuous bed (monolithic) technique as an attractive alternative for the classical packed columns in chromatography, stimulated further investigations in this direction. The research data published since that time on the development and application of the continuous beds formed using hydrophobic interaction-based phase separation mechanism are reviewed. Some innovative species of the beds, such as polyrotaxane beds or nonparticulate restricted-access materials for direct analysis of the biological fluids in the capillary format are also discussed. Characteristic features and practical details of the continuous bed technique are revealed. Due to many advantages, the continuous bed technique became a competitor with the traditional packings in capillary or chip-based microanalysis. The importance of the continuous bed morphology on the chromatographic characteristics is shown. The applicability of modern microscopic analysis to evaluate the morphology of the continuous beds is demonstrated.
Chromatography, Liquid↗
Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography.
There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.
Benzene Derivatives↗
Novel assay format permitting the prolonged use of regeneration-based sensor chip technology.
A polyclonal antibody raised against morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine) was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor. Previously developed assays have generated calibration curves based on differences in the quantity of response units binding to the surface of a chip coated with the analyte. The novel assay described here was based on the development of a standard curve using the slope of a series of consecutive binding interactions. Using this format, regeneration between each assay cycle was no longer required. This increased the useable life span of the chip surface and, as a result, decreased the cost associated with the assay. Thus, at least 15 binding interactions could be carried out before the saturation of antibody on the surface of the chip caused the response to deviate significantly from linearity. After 15 nonregenerated binding interactions, the slope still remained within 1.5% of the slope after a single binding event. Analysis time, and the sample volumes required were also markedly decreased while sensitivity was enhanced. The inhibition assay developed had a detection range of 270 to 17,500 pg ml(-1).
Antibodies↗