Microfluidic and transducer technologies for lab on a chip applications.

Point-of-care diagnostic devices typically require six distinct qualities: they must deliver at least the same sensitivity and selectivity, and for a cost per assay no greater than that of today's central lab technologies, deliver results in a short period of time (〈15 min at GP; 〈2h in hospital), be portable or at least small in scale, and require no or extremely little sample preparation. State-of-the-art devices deliver information of several markers in the same measurement.

A packaged optical slot-waveguide ring resonator sensor array for multiplex label-free assays in labs-on-chips.

We present the design, fabrication, and characterisation of an array of optical slot-waveguide ring resonator sensors, integrated with microfluidic sample handling in a compact cartridge, for multiplexed real-time label-free biosensing. Multiplexing not only enables high throughput, but also provides reference channels for drift compensation and control experiments. Our use of alignment tolerant surface gratings to couple light into the optical chip enables quick replacement of cartridges in the read-out instrument. Furthermore, our novel use of a dual surface-energy adhesive film to bond a hard plastic shell directly to the PDMS microfluidic network allows for fast and leak-tight assembly of compact cartridges with tightly spaced fluidic interconnects. The high sensitivity of the slot-waveguide resonators, combined with on-chip referencing and physical modelling, yields a volume refractive index detection limit of 5 x 10(-6) refractive index units (RIUs) and a surface mass density detection limit of 0.9 pg mm(-2), to our knowledge the best reported values for integrated planar ring resonators.

A quantitative study of optical mapping surfaces by atomic force microscopy and restriction endonuclease digestion assays.

Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.

Investigation of protein patterns in mammalian cells and culture supernatants by matrix-assisted laser desorption/ionization mass spectrometry.

The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states. Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization. Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological systems. Several mammalian cell lines including CHO DXB11, CHOSSF3, and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions. Initial experiments to observe the success of protein translation in gene expression experiments were performed. Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16,000 to 75,000 Da. In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS-PAGE electrophoresis experiments. These new methods were used to monitor a large-scale cultivation of hybridoma cells expressing an antibody of the IgG type. The increase in whole antibody and antibody light-chain protein, 8650 Da, and the decrease of insulin were followed during the monitoring period. Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC.

Influence of solvents and detergents on matrix-assisted laser desorption/ionization mass spectrometry measurements of proteins and oligonucleotides.

The effect of solvents was found to be critical for sample preparation in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For proteins and oligonucleotides the use of 2-propanol/water as a solvent for different matrices can significantly improve the quality of spectra. This effect is demonstrated with proteins ranging in molecular weight from 12 to 150 kDa and with a special 19-mer oligonucleotide. A comparison of MALDI-MS using of 2-propanol as matrix solvent and high-performance capillary electrophoresis resulted in identical relative peak intensities for a p(dT)12-18 oligonucleotide mixture. Additionally, the effect of detergents for characterization of high molecular weight proteins in very dilute solutions was studied with this solvent. It was found that Triton X-100, up to a concentration of 1%, was highly compatible with MALDI measurements and even could improve the quality of spectra. Use of detergents for cell profiling has extended the detectable mass range to about m/z 75,000.

Analysis of drug/plasma protein interactions by means of asymmetrical flow field-flow fractionation.

PURPOSE: The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. METHODS: Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. RESULTS: Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. CONCLUSIONS: If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.

Evaluation of the cell specificity and sulfate dependence of glomerular extracellular matrix proteoglycan synthesis.

Homogeneous cultures of epithelial, endothelial, and mesangial cells from calf glomeruli were radiolabeled with [35S]sulfate in order to evaluate their capacity for the biosynthesis of the proteoglycan (PG) components present in the glomerular extracellular matrix. Although each cell type was observed to incorporate into its matrix predominantly immunologically related heparan sulfate (HS) PGs (M(r) approximately 500 kDa), endothelial and mesangial cells also deposited substantial amounts of PGs with chondroitin sulfate (CS) and dermatan sulfate (DS) chains. The limited capacity of epithelial cells to synthesize PGs other than those containing HS was also evident from the immunologically distinct components (M(r) approximately 300 kDa) shed into the medium which in contrast to those from the endothelial and mesangial cells contained no CS and only small amounts of DS glycosaminoglycans. While the matrix proteoglycan HS chains differed in length depending on cell type, they were similar in containing the six mono- and disulfated disaccharide species previously found in bovine glomerular basement membrane, including the distinctive iduronic-GlcNSO3 (3-SO4) sequences. While the addition of sulfate to medium free of this ion brought about no change in HS PG production by any of the three cell types and the formation of CS and DS chains by epithelial and mesangial cells was unaffected, the formation of CS/DS PGs by endothelial cells was altered to a pronounced extent through the conversion of an undersulfated PG to a more polyanionic molecule. Our findings are consistent with the concept that the glomerular extracellular matrix is made up of two biosynthetically distinct regions (mesangium and basement membrane) and are relevant to an understanding of various diseases affecting the renal filter.

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha.

A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The recombinant GP Ib alpha fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 micrograms of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib alpha fragment.

Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.

The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.

Preparation and mass spectral characterization of pentafluorobenzyl derivatives of alkyl and hydroxyalkyl-nucleobase DNA adducts.

Pentafluorobenzyl (PFBz) derivatives of the following nucleobases were prepared: cytosine, 5-methylcytosine, O2-methylcytosine, O2-ethylthymine, O4-ethylthymine, 5-hydroxymethyluracil, N6-methyladenine, O6-methylguanine, O6-hydroxyethylguanine and O6-hydroxyethylpurine. 13C nuclear magnetic resonance was diagnostic for O- versus N-attachment of the PFBz moiety: the resonance of the methylene carbon appeared in the range 29.15-42.13 ppm for NCH2C6F5, and 58.45-69.01 for OCH2C6F5. Considerable structural information was provided by mass spectrometry with ionization by electron impact. All of the derivatives were detected with high sensitivity and specificity by gas chromatography with detection by electron capture negative ion mass spectrometry, reflecting not only their chemical and physical stability, but also their strong tendency to form a structurally diagnostic anion, [M - PFBz]-, in high yield under these ionization conditions. PFBz derivatives are therefore attractive forms of alkyl-substituted nucleobases for analysis by mass spectrometry.

Ester and related derivatives of ring N-pentafluorobenzylated 5-hydroxymethyluracil. Hydrolytic stability, mass spectral properties, and trace detection by gas chromatography-electron-capture detection, gas chromatography-electron-capture negative ion mass spectrometry, and moving-belt liquid chromatography-electron-capture negative ion mass spectrometry.

One consequence of radiation damage to DNA is the conversion of thymine to 5-hydroxymethyluracil (HMU). In order to sensitively detect this DNA adduct by gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electron-capture detection techniques, it is necessary to derivatize it. This study was designed to select an optimum ester derivative of the aliphatic hydroxyl group on HMU. N1, N3-Bis(pentafluorobenzyl)-HMU was formed as a parent derivative, and from this a series of esters. Also O-pentafluorobenzyl and O-tetrafluorobenzyl ether derivatives were prepared. Of the esters the pivalyl derivative was the best choice because it formed easily, was relatively stable to aqueous hydrolysis (t 1/2 = 9.8 days at pH 11.5, 24 degrees C) and gave a response at fmol levels by GC and LC comparable to that of the ethers. Unanticipated was a good response as well for the parent derivative, a free hydroxyl compound, by GC and LC at this level. The work also demonstrates a high performance by LC-electron-capture negative ion mass spectrometry with a belt interface for the trace detection of derivatives of this type.

Trace detection of modified DNA bases via moving-belt liquid chromatography-mass spectrometry using electrophoric derivatization and negative chemical ionization.

Electrophoric derivatives of 5-methylcytosine and 5-hydroxymethyluracil nucleobases are determined using high-performance liquid chromatography-mass spectrometry coupled via a moving-belt interface. Standards as well as samples derived from DNA are analysed. As little as 9.9 pg (signal-to-noise ratio 5) and 180 fg (signal-to-noise ratio 10) of the respective nucleobases are detected in the electron-capture negative chemical ionization mode, and linear responses are observed over a moderate dynamic range. In a comparison study, liquid chromatography-electron-capture negative chemical ionization mass spectrometry is found to have a sensitivity comparable to gas chromatography-electron-capture negative chemical ionization mass spectrometry for 5-hydroxymethyluracil. A detection limit of 60 fg (signal-to-noise ratio 5) by gas chromatography-mass spectrometry is only three-fold better than the amount detected by liquid chromatography-mass spectrometry using the same mass spectrometer.

Pentafluorobenzylation of alkyl and related DNA base adducts facilitates their determination by electrophore detection.

The DNA adduct O4-ethylthymine can be alkylated under mild conditions with pentafluorobenzyl bromide. The product has good gas chromatographic characteristics and also forms a structurally characteristic anion in high yield when subjected to electron capture mass spectrometry. Related adducts for other DNA bases behave similarly. These properties stimulated us to develop a general analytical method based on the derivatization reaction. Important for this method is an oxidation-elimination reaction that mildly releases a base from a nucleoside. Thus, a general analytical method based on pentafluorobenzylation is now available for determining many alkyl and related DNA adducts.