Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography.

We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and ß-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.

Solution mixing and the emission of light in flow-cells for chemiluminescence detection.

Constructing flow-through reactors for chemiluminescence detection by machining channels into polymer disks has enabled the exploration of new configurations and materials that can improve signal intensity beyond that attainable with the traditional coiled-tubing design. Several approaches to merge reactant solutions were examined: an intersection, chamber or deeper well in the centre of a serpentine configuration flow-cell (directly in front of a photomultiplier tube), or a confluence point outside the detection zone. For several analytically useful, rapid chemiluminescence reactions, the single-inlet flow-cell with external Y-piece was most suitable, but for others (such as KMnO(4)/Mn(II) with morphine, and [Ir(f-ppy)(2)BPS](-) with fluoroquinolones) the dual-inlet configuration provided greater signals. The introduction of central mixing zones with larger widths than the channel reduced the chemiluminescence response. The reversing turns of a serpentine channel promote efficient mixing and greater chemiluminescence intensities than a spiral channel, but increasing the sharpness of the turns created areas of poor solution flow and decreased the chemiluminescence response. Teflon disks impregnated with glass microspheres increased the chemiluminescence signals by 13%-17%, due to the greater reflection of stray light towards the photodetector.

Autocatalytic nature of permanganate oxidations exploited for highly sensitive chemiluminescence detection.

Manganese(II) salts catalyze the chemiluminescent oxidation of organic compounds with acidic potassium permanganate. The formation of insoluble manganese(IV) species from the reaction between manganese(II) and permanganate can be prevented with sodium polyphosphate, and therefore, relatively high concentrations of the catalyst can be added to the reagent before the light-producing reaction is initiated. The rapid and intense emissions from these manganese(II) catalyzed chemiluminescence reactions provide highly sensitive detection and greater compatibility with liquid chromatography.

Precision milled flow-cells for chemiluminescence detection.

Novel flow-cells with integrated confluence points and reaction channels designed for efficient mixing of fast chemiluminescence systems were constructed by machining opposing sides of a polymer chip and sealing the channels with transparent epoxy-acetate films. A hole drilled through the chip provided the conduit from the confluence point on one side to the centre of the reaction zone on the other side, allowing rapid presentation of the reacting mixture to the photodetector. The effectiveness of each flow-cell was evaluated by comparing the chemiluminescence intensity using flow injection analysis methodology, and examining the distribution of light emanating from the reaction zone (captured by photography in a dark room) when the reactants were continuously merged. Although previously reported chemiluminescence detectors constructed by machining channels into polymers have almost exclusively been prepared using transparent materials, we obtained far greater emission intensities using an opaque white chip with a thin transparent seal, which minimised the loss of light through surfaces not exposed to the photomultiplier tube. Furthermore, this approach enabled the exploration of reactor designs that could not be incorporated in traditional coiled-tubing flow-cells.

Chemiluminescence detector with a serpentine flow cell.

We present a new chemiluminescence detector, with solution channels that have been machined into a Teflon disk and sealed with a sapphire window. The configuration of the flow cell can be conveniently modified by replacing the Teflon disk. A comparison of some existing and novel designs, using the chemiluminescence reaction of morphine with acidic potassium permanganate and the bioluminescence reaction of ATP with the commercially available "BacTiter-Glo" reagent, has revealed that a serpentine channel allows greater quantities of light to be captured than a spiral channel, due to more efficient mixing of the analyte and reagent solutions within the cell.

Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics.

BACKGROUND: Measuring plasma unbound bilirubin concentration by the peroxidase test is useful in the management of jaundiced newborns. However, the commercially available peroxidase technology is manual, and the unbound bilirubin may be seriously underestimated at the 42-fold sample dilution and single peroxidase concentration used. We investigated improving the test by adapting it to Zone Fluidics, which is a system for automating reactant handling that requires small sample volumes and dilution. METHODS: A computer-directed Zone Fluidics system was constructed using small diameter tubing to connect in series a water-surfactant reservoir, a bi-directional pump, a multiport selection valve to which peroxidase test reactants (45 mul of sample) are attached with one port open to air, and a spectrophotometer flow cell. Test reactants and air are sequentially aspirated through the valve into the tubing connecting the pump and valve to form a reactant "zone" surrounded by air. The zone is advanced to the spectrophotometer flow cell where total and unbound bilirubin are determined (37 degrees C) from the absorbance at 460 nm at a 2-fold sample dilution and 4 peroxidase concentrations. Imprecision was assessed in artificial controls and newborn plasma. Plasma results were compared with those obtained using the commercial method. RESULTS: The CV for unbound bilirubin in the various controls ranged from 11% to 38% (within day) and 12% to 27% (between days). Triplicate CV measurements for newborn plasma measurements ranged from 0.6% to 31% (mean 11%, n=47). Mean unbound bilirubin by Zone Fluidics was 5-fold higher than that by the commercial method. CONCLUSION: Zone Fluidics can be used to automate the peroxidase test and overcome many of the limitations of the commercially available peroxidase technology.

Development of an automated sample preparation module for environmental monitoring of biowarfare agents.

An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.