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.
A rapid, automated, microanalytical procedure for the determination of 90 Sr in aged nuclear waste has been developed. It is based on a sequential injection analysis (SIA) system which rapidly separates 90 Sr from 90 Y, 137 Cs, and other radionuclides. The isolated 90 Sr is then detected on-line with a flow-through liquid scintillation counter. The separation is achieved using a sorbent extraction minicolumn containing a resin (EIChrom, Sr-Spec) that selectively binds 90 Sr as a crown ether complex under acidic conditions. The 90 Sr is eluted with water, mixed with liquid scintillation cocktail, and detected in the flow cell of the counter. Sample 90 Sr activity can be quantified from peak areas, giving linear calibration curves. The instrument can also be operated in a stoppedflow mode for longer counting times. Analyses of aged nuclear waste samples from the Hanford site by the SIA method and a manual method were in excellent agreement: correlation coefficient R ) 0.994. Sample analysis by the SIA method is complete in less than 40 min. The advantages of the new 90 Sr analyzer include faster analysis, greater precision, reduced labor costs, and reduced secondary waste. Worker safety is improved because solution handling operations are fully automated and contained.
Sodium borohydride-based hydride generation was automated by using programmable flow within the lab-on-valve module. Mercury vapor, generated in the reaction mixture, was extracted in a gas/liquid separator. The gas-expansion separator was miniaturized and compared with the performance of a novel gas separator that exploits the combination of Venturi effect and reduced pressure. Cold vapor atomic spectroscopy was used as a model system, with detection of mercury by absorption at 254 nm and limit of detection of 9 microg of Hg/L, using 300 microL of sample and 100 microL of borohydride. This work introduces, for the first time, sequential injection technique for hydride generation, highlights advantages of using programmable flow, and outlines means for miniaturization of assays based on spectroscopy of volatile species.
A rational design of the sequential injection analyzer is based on description of the mutual penetration of sample and reagent zones, which are sequentially stacked into a tubular conduit and then injected into a reactor and transported toward the detector by means of a carrier stream, flowing at a preprogrammed rate. Variables governing zone penetration have been identified by a series of dispersion experiments, for sequential injection of two and three zones, thus outlining the conditions for performing single- and double-reagent-based assays. A parallel is drawn between conventional flow injection and corresponding sequential injection colorimetric determinations of chloride and phosphate, with the aim of suggesting guidelines for the development of spectrophotometric- and fluorescence-based sequential injection methods.
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.
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