Search PubMed⌕ Search

Biomedical subjects

J Ruzicka

Publications and source records attributed to J Ruzicka.

At least 19 recordsLinked to original sources
PubMed2001

Micro sequential injection: fermentation monitoring of ammonia, glycerol, glucose, and free iron using the novel lab-on-valve system.

Using an integrated lab-on-valve manifold in a microfluidic sequential injection format (microSI), automated sample processing has been developed for off-line and on-line monitoring of small-scale fermentations. Spectrophotometric assays of ammonia, glucose, glycerol, and free iron were downscaled to use micro-quantities of commercial reagents. By monitoring the reaction rate, the response curves in a stopped-flow mode generate linear calibration curves for ammonia [r2 = 1.000 (0.9% SE)], glycerol [r2 = 0.999 (1.1% SE)], glucose [r2 = 0.999 (1.1% SE)], and free iron [r2 = 0.999 (1.5% SE)]. Since sample dilution and reagent quantities are easily adjusted within the programmable SI format, the lab-on-valve system can accommodate samples over a wide concentration range (ammonia: 3-1200 ppm; glycerol: 20-120 ppm; glucose: 35-1000 ppm; and free iron: 80-400 ppm). This work demonstrates the key advantages of miniaturization through the reduction of sample and reagent use, minimizing waste and providing a compact yet reliable instrument. The lab-on-valve manifold uses a universal hardware configuration for all analyses, only requiring changes in software protocol and choice of reagents. All of these features are of particular importance to small-scale experimental fermentation where multiple analyte analyses are needed in real-time using small sample volumes. It is hoped that this first real-life application of the lab-on-valve manifold will serve not only as a model system to downscale assays in a practical fashion, but will also inspire and promote the use of the integrated microSI manifold approach for a wider range of biotechnological applications.

Journal Article↗
PubMed2001

Micro sequential injection: environmental monitoring of nitrogen and phosphate in water using a "Lab-on-Valve" system furnished with a microcolumn.

A "Lab-on-Valve" manifold operated in the micro sequential injection (microSI) mode was adopted to accommodate EPA-approved methods for spectrophotometric determinations of nitrate, nitrite and orthophosphate in the ppb (N or P) concentration range. A computer programmable microSI protocol, utilizing stopped-flow within a copperized Cd-foil filled microcolumn was developed for nitrate reduction to nitrite with subsequent colorimetric measurement, yielding concentration ranges for nitrate of 100.0-4000.0 ppb (N) and for nitrite of 30.0-4000.0 ppb (N) and linear calibration responses of r2 = 0.9999 for nitrate and 0.9995 for nitrite. Using a stopped-flow reaction rate measurement, phosphate was determined in the range 1.0-30.0 ppb (P) with a calibration response of r2 = 0.9997. The technical improvement of this methodology, apart from micro miniaturization, is the use of the stopped-flow technique, that resulted in improved detection limits and allowed reagent consumption to be reduced 1500-fold compared with conventional procedure while the amount of metallic cadmium was reduced 20-fold compared with the EPA-approved continuous-flow assay.

Flow Injection Analysis↗
PubMed2001

Effects of ultrafiltration, dialysis, and temperature on gas exchange during hemodiafiltration: a laboratory experiment.

To study gas exchange in the filter during continuous venovenous hemodiafiltration (CVVHDF), an air-tight heated mixing chamber with adjustable CO2 supply was constructed and connected to a CVVHDF monitor. Bicarbonate-free crystalloid (Part 1) and packed red blood cell (Part 2) solutions were circulated at 150 ml x min(-1). Gas exchange expressed as pre-postfilter difference in CO2 and O2 contents was measured at different CVVHDF settings and temperatures of circulating and dialysis solutions. Ultrafiltration was most efficacious for CO2 removal (at 1,000 ml x h(-1) ultrafiltration CO2 losses reached 13% of prefilter CO2 content). Addition of dialysis (1,000 ml x h(-1)) increased CO2 loss to 17% and at maximal parameters (filtration 3,000 ml x h(-1), dialysis 2,500 ml x h(-1)), the loss of CO2 amounted to 35% of prefilter content. Temperature changes of circulating and/or dialysis fluids had no significant impact on CO2 losses. The O2 exchange during CVVHDF was negligible. Currently used CVVHDF is only marginally effective in CO2 removal. Higher volume ultrafiltration combined with dialysis can be expected to reach clinical significance.

Carbon Dioxide↗
PubMed2000

Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy.

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

Animals↗
PubMed1999

Detection of oxygen consumption of cultured adherent cells by bead injection spectroscopy.

This paper describes a method for detecting oxygen consumption of adherent cell cultures. The sensing is based on oxygen-dependent quenching of the phosphorescence of a Pt-porphyrin complex immobilized on microcarrier beads, which are used as the cell culture substrate. Bead injection, a recent variant of the flow injection technique, is used to pack an aliquot of the beads into a small sensing layer that can be easily and rapidly renewed. The technique is tested on a model system of Chinese Hamster Ovary M1 cells grown on Cytodex-3 microcarrier beads. Cellular respiration is monitored through O2 consumption measured across a period of 3 min. The method is validated by detecting the impairment of aerobic metabolism caused by 1.5 mM amobarbital. Further, it is shown to have enough precision to distinguish even more subtle changes, such as the increase in oxygen consumption caused by stimulation of the muscarinic m1 receptor with 100 microM carbachol.

Animals↗
PubMed1999

Flow injection microscopy for the study of intracellular calcium mobilization by muscarinic agonists.

The study of cellular response to chemical agonists is essential in understanding the complex functions mediated by cell surface receptors. Flow injection microscopy has been used with the CHO-M1-WT3 cell line and the fluorescent Ca2+ indicator Fura-2-AM to monitor mobilization of internal Ca2+. Repeated stimulation of cells mounted in an inverted radial flow chamber allows the direct comparison of relative intracellular Ca2+ mobilization with respect to agonist dose. The process of determining dose-response relationships is simplified since an entire dose-response curve can be constructed from a distinct set of cells. Use of flow injection lends precision to the application and removal of agonists while allowing cellular activity to be monitored throughout the stimulation and recovery processes. In this work, dose-response curves have been constructed for the muscarinic agonists carbachol, acetylcholine, and pilocarpine resulting in EC50 values of 1.7 microM, 56 nM, and 6.8 microM, respectively.

Acetylcholine↗
PubMed1999

A flow injection renewable surface technique for cell-based drug discovery functional assays.

A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-à-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.

Animals↗
PubMed1999

Automation of functional assays by flow injection fluorescence microscopy.

Bead-injection spectroscopy is a novel technique that uses immobilized eukaryotic cells on microbeads as a renewable biosensor for fluorescence microscopy. The use of a flow injection instrument allows fast functional assays that generate full kinetic characterization of a drug. Because the cell population is automatically replaced for each assay, variability is minimized, thus allowing greater accuracy.

Biotechnology↗
PubMed1997

Bioligand interaction assay by flow injection absorptiometry.

A novel technique for the study of bioligand interactions based on combining flow injection on renewable surfaces with UV-visible absorptiometry is introduced. The concept is proven by monitoring the binding of various proteins to protein G and the binding of various insulin analogs to a monoclonal anti-insulin antibody, thus providing a comparison with the performance of surface plasmon resonance (SPR)-based techniques. The advantages of the bioligand interaction flow injection absorptiometry approach are speed, low cost, no need for regeneration of solid substrate, and spectral resolution not available with SPR sensing. It is believed that this technique will have an impact on the entire biosensor field since it allows simultaneous monitoring of labeled as well as nonlabeled species in real time over a wide spectral range with high sensitivity.

Adsorption↗
PubMed1997

Flow injection renewable surface immunoassay for real time monitoring of biospecific interactions.

An automated system for performing rapid immunoassay, kinetic measurements, and affinity ranking of biomolecular interactions using fluorescence-labeled ligands is described. Its distinctive feature is the automated renewal of solid phase for each measurement, which avoids the need for regeneration of the sensing surface. This system--flow injection renewable surface immunoassay (FIRSI)--is used for the first time here for determination of rate constants for an antibody/antigen interaction and for affinity ranking of several related antigens against one antibody. The performance of the system is compared with a commercial BIAcore system that uses surface plasmon resonance for monitoring biomolecular interactions. While the values of association and dissociation rate constants for human serum albumin (HSA) with monoclonal anti-HSA antibody obtained by these techniques were comparable, it is shown that the FIRSI techniques requires simpler instrumentation, handles a broader size range of analytes, and does not suffer from disturbances caused by changes in the refractive index.

Animals↗
PubMed1997

Microfabricated flow chamber for fluorescence-based chemistries and stopped-flow injection cytometry.

A microfabricated flow chamber (MFC) suitable for performing liquid-based fluorimetric assays is introduced. Precision delivery of microliter volumes of sample and reagent to the MFC is accomplished by a double-syringe-pump flow injection analysis (FIA) apparatus. The FIA-MFC system also combines the 'sheath flow' technique (traditionally used in flow cytometry) and stopped-flow FIA as a way to allow sample and reagent streams to be mixed reproducibly. The applicability of this FIA-MFC system to bioanalytical assays is demonstrated by performing an enzymatic assay with an artificial fluorigenic substrate to determine the activity of Savinase, a proteolytic enzyme. When coupled to a fluorescence microscope platform, quantitative analysis of the reaction product is possible. Experiments showed that the FIA-MFC system was capable of performing the assay with good reproducibility of injection (1.5%), and linearity of response (r2 = 0.9997) in activity ranges of analytical interest. Owing to the incorporation of flow cytometry sheath flow principles into an FIA format, the FIA-MFC system is a suitable tool for cytometric studies.

Detergents↗
PubMed1996

Coaxial flow mixer for real-time monitoring of cellular responses in flow injection cytometry.

Improved time resolution of kinetic cellular events in flow cytometry is demonstrated by using a coaxial flow-mixing device integrated within a flow-injection (FI) system. The instrument is used in combination with a Becton Dickinson FACS Analyzer for on-line reagent addition, rapid sample mixing, and temperature control of cell suspensions. The coaxial flow device can instantaneously (< 60 ms) mix reagent and sample streams, allowing cytometric analysis of subsecond events to be performed. Kinetic measurements can be performed on the FACS analyzer in a variable time range of from 100 ms to 3 min. The system also allows the collection of unlimited cellular events at a specific incubation time point. Because the system operates continuously and no boost in core flow is required, disturbances of flow conditions are avoided. The capabilities of the flow injection cytometer have been demonstrated by the determination of internal [Ca2+]i mobilization in Jurkat T lymphocytes perfused internally with INDO-1 and stimulated by ionomycin.

Flow Cytometry↗
PubMed1996

Flow injection microscopy: a novel tool for the study of cellular response and drug discovery.

Studying responses of live cells to agonists, antagonists and other physical stimuli offers insight into their complex membrane and internal biochemistry. An experimental technique has been developed in which responses of living cells in an inverted radial flow chamber are continuously monitored while being repeatedly stimulated using controlled pulses of a biologically active ligand. Precisely defined flow conditions result in reproducible peaks which can be numerically analysed by comparison with a tracer curve obtained by substituting a dye for the stimulus. Exploratory studies have demonstrated that the flow injection technique can provide a novel method for kinetics of receptor binding and cellular responses. Flow injection microscopy (FIM) allows identification of biologically active ligands and their ranking based on measurement of the cellular responses in a short time frame. The use of FIM for rapid drug screening, through monitoring of the initial kinetics of cellular responses, is demonstrated on a model system.

Cell Line↗
PubMed1995

A coaxial jet mixer for rapid kinetic analysis in flow injection and flow injection cytometry.

A simple coaxial jet mixer for rapid and efficient confluent mixing under laminar flow conditions (Re < 5) is described. This device demonstrates exceptional control of mixing between two laminar streams by creating shear forces due to variable flow velocities at the point of confluence. It is suitable for flow injection and cytometric analyses of rapid kinetic events which require contact mixing of two solutions and subsecond measurements of the evolving reaction. This apparatus was devised for flow injection cytometry as performed on a Becton Dickinson FACS Analyzer. Under normal cytometric conditions and at a sample introduction rate of 60 microL/min, the laminar jet mixer is capable of complete mixing of two solutions within 55 ms. Kinetic measurements can be performed on the FACS Analyzer in a variable time range of 100 ms to 3 min with 14-30 ms temporal resolution of the studied event. Since no boost in core flow is required, potential spectral distortions due to core flow variations are eliminated. This coaxial jet mixer can be easily constructed and employed on a variety of cytometers as well as conventional flow injection analysis systems, since it is an effective mixer under most flow conditions.

Flow Cytometry↗
PubMed1995

Configurations of a flow injection system for perfusion studies of adherent cells.

This paper describes three configurations of a flow injection apparatus designed to be the fluidic drive for perfusion studies of cultured adherent cells. The apparatus was coupled to a flow-through perfusion chamber that was specifically designed for live cell perfusion using fluorescence microscopy as the detector. The instrument consists of two linear syringe pumps and a multiposition selector valve which, under computer control, allowed sequential injection, fluid switching, and flow injection to be performed with minimal system reconfiguration. When the apparatus is coupled to a dual inlet perfusion chamber, target cells can be exposed to very steep reagent pulses, while the traditional single inlet perfusion chamber allows more flexibility and provides a more gradual increase in reagent concentration. The most significant salient feature of the system is the ability to generate very steep pulses--a desirable feature for cell perfusion studies.

Cell Adhesion↗