Jaromir Ruzicka

Sequential injection : a new concept for chemical sensors, process analysis and laboratory assays

Abstract Established flow-injection techniques allow advanced solution handling for laboratory purposes, but chemical sensing and continuous monitoring of chemical processes require dramatically simplified flow schemes and instrumentation with the potential for miniaturization and an inherent ruggedness. Considerations based on the random walk model have led to the concept of sensor injection and sequential injection analysis. This new approach to automated analysis is designed to fill a gap in present flow-injection methodology.

Lab-on-valve: universal microflow analyzer based on sequential and bead injection

This paper introduces a novel methodology for downscaling reagent based assays to micro- and submicroliter level. It is shown that sample handling in the sequential injection mode, which employs forward, reversed and stopped flow, can be programmed to accommodate a wide variety of assays within the same microfluidic device. Solution metering, mixing, dilution, incubation and monitoring can be executed in any desired sequence in a system of channels, integrated with a multipurpose flow cell. The channel system and flow cell are fabricated as a monolithic structure mounted atop a conventional multiposition valve. In addition to compactness, the advantage of this ‘lab-on-valve’ system is the permanent rigid position of the sample processing channels that ensures repeatability of microfluidic manipulations, controlled by conventional sized peripherals. With the exception of the integrated microconduit system, that has been designed and mesofabricated by computer aided design (CAD) technology, all peripherals (sequential injection system, fiber optic UV/VIS spectrophotometer-fluorometer) are conventional sized and commercially available components. This provides proven robustness and reliability of operation, and makes the microfluidic system compatible with real life samples and peripheral instruments. The system has been characterized by dye injection, to provide guidelines for method development. Its versatility is documented by a phosphate assay, enzymatic activity assay of protease and by a bioligand interaction assay of immunoglobulin G (IgG) based on its interaction with protein G immobilized on Sepharose beads.

Sorbent extraction in flow injection analysis and its application to enhancement of atomic spectrometry

Abstract The extraction of metals as their chelates from aqueous samples can be simplified, miniaturized and automated by flow injection/sorbent extraction techniques. The chelate is formed in the flow stream, sorbed on C-18 bonded silica and then eluted for transfer to the atomic absorption spectrometer. The proposed method is shown to be useful for the preconcentration of copper and lead by means of their chelation with diethyldithiocarbamate or 8-quinolinol; complexation with 1-(2- pyridylazo)-2-naphthol or 4-(2-pyridylazo)resorcinol is also possible. This approach enables organic reagents to be used directly, without immobilization, in “open” flow-injection systems for preconcentration or separation of analytes.

Sequential injection wetting film extraction applied to the spectrophotometric determination of chromium(VI) and chromium(III) in water

The spectrophotometric determination of Cr(VI) and Cr(III) via sequential injection was used to demonstrate the sensitivity enhancement provided by a newly developed wetting film extraction system. The reaction product of Cr(VI) with 1,5-diphenylcarbazide was ion-paired with perchlorate and extracted into an organic wetting film consisting of octanol and 4-methyl-2-pentanone on the inner wall of a Teflon tube. The wetting film, with the extracted analyte, was then eluted with 100 mul acetonitrile and the analyte determined spectrophotometrically at 546 nm. Important optimized parameters were the selection of wetting film and elution solvents, the flow rate, the length and diameter of the extraction coil and the conditions for the formation of the ion paired chelate. Cr(III) was previously oxidized to Cr(VI) and calculated as the difference between total Cr and Cr(VI). An enrichment factor of 25 and a detection limit of 2.0 mug l(-1) Cr(VI) were achieved with a sampling frequency of 17 h(-1). The calibration curve was linear up to 100 mug l(-1) Cr(VI) (r = 0.999). The relative standard deviations were 2.8 and 2.0% at the 25 and 100 mug l(-1) levels.

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose, Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.

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.

Flow injection based renewable electrochemical sensor system.

A novel class of electrochemical sensors is proposed utilizing electrically conducting beads to form a disposable electrode as well as nonconducting beads to form renewable layers of immobilized enzymes. The concept, aimed to prevent fouling, is tested on an amperometric sensor coupled to nonconducting beads with different immobilized oxidases:  galactose, lactate, alcohol, or glucose oxidase, the latter two being used to determine alcohol and gluocse, respectively, in samples of beer and wine. Glucose oxidase was also immobilized on conducting glassy carbon particles to explore the performance of a biosensor where both enzyme and electrode can be automatically renewed in less than 1 min. The results confirm that the concept of a flow injection renewable electrochemical sensor (FI-RES) is practical. It provides a novel approach to biosensing, to comparing enzyme activity, to studying enzyme immobilization on different supports, and to voltammetry in general.

Sequential Injection Separation System with Stopped-Flow Radiometric Detection for Automated Analysis of 99Tc in Nuclear Waste

An automated procedure for the determination of (99)Tc in aged nuclear waste has been developed. Using advanced sequential injection (SI) analysis instrumentation, (99)Tc(VII) is separated from radioactive and stable interferences using a TEVA resin column that selectively retains pertechnetate ion from dilute nitric acid solutions. The separated (99)Tc is eluted with 6 M nitric acid and quantified on-line with a flow-through liquid scintillation detector. A stopped-flow technique has been optimized that improves the analysis precision and detection limit compared to continuous-flow detection, reduces consumption of liquid scintillation cocktail, and increases sample throughput by separating the next sample while the present sample is being counted. The detection limit is 30 pCi, or 2 ng, of (99)Tc, using a 15-min stopped-flow period. The analysis time is 40 min for the first sample and is reduced to 20 min for each subsequent sample. Processed nuclear waste samples from the Hanford site were successfully analyzed by this new method.

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.

Homogeneous and heterogeneous systems : Flow injection analysis today and tomorrow

Abstract Chemical conversions by means of soluble and immobilized reagents including catalysts (enzymes) are reviewed, and estimates are made towards development of miniaturized devices which besides handling homogeneous samples will have the capability to process heterogeneous sample materials such as blood. Reference covering available FIA literature published in 1987 are listed in an Appendix.