Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescence determinations

A three-layer poly(dimethylsiloxane) (PDMS)/glass microfluidic system for performing on-chip solid-phase enzymatic reaction and chemiluminescence (CL) reaction was used for the determination of glucose as a model analyte. A novel method for the immobilization of controlled-pore-glass based reactive particles on PDMS microreactor beds was developed, producing an on-chip solid-phase reactor that featured large reactive surface and low flow impedance. Efficient mixing of reagent/sample/carrier streams was achieved by incorporating chaotic mixer structures in the microfluidic channels. A conventional sequential injection (SI) system was adapted for direct coupling with the microfluidic system, and combined with hydrostatic delivery of reagents to achieve efficient and reproducible sample introduction at 10 μl levels. A detection limit of 10 μM glucose (3σ), and a precision of 3.1% RSD (n=7, 0.2 mM glucose) were obtained using the SI-microfluidic-CL system integrated with a glucose oxidase (GOD) reactor. Carryover was <5% at a throughput of 20 samples/h.     

Coupling continuous ultrasound-assisted extraction, preconcentration and flame atomic absorption spectrometric detection for the determination of cadmium and lead in mussel samples

Continuous ultrasound-assisted extraction has been coupled with preconcentration and flame atomic absorption spectrometry for the determination of cadmium and lead in mussel samples. Experimental designs were used for the optimisation of the leaching and preconcentration steps. The use of diluted nitric acid as extractant in the continuous mode at a flow rate of 3.5 ml min⁻¹ and room temperature was sufficient for quantitative extraction of these trace metals. A minicolumn containing a chelating resin (Chelite P, with aminomethylphosphoric acid groups) was proved as an excellent material for the quantitative preconcentration of cadmium and lead prior to their flame atomic absorption detection. A flow injection manifold was used as interface for coupling the three analytical steps, which allowed the automation of the whole analytical process. A good precision of the whole procedure (2.0 and 2.3%), high enrichment factors (20.5 and 11.8) and a detection limit of 0.011 and 0.25 μg g⁻¹ for cadmium and lead, respectively, were obtained for 80 mg of sample. The sample throughputs were ca. 16 and 14 samples h⁻¹ for cadmium and lead, respectively. The accuracy of the analytical procedures was verified by using a standard reference material (BCR 278-R, mussel tissue) and the results were in good agreement with the certified values. The method was successfully applied to the determination of trace amounts of cadmium and lead in mussel samples from the coast of Galicia (NW, Spain).     

Electrochemical determination of iodide on a vanadium oxide-polypropylene carbonate coated glassy carbon electrode

A vanadium oxide-modified glassy carbon electrode was simply and conveniently fabricated by casting vanadium tri(isoproxide) oxide (VO(OC₃H₇)₃) and poly(propylene carbonate) (PPC) onto the glassy carbon electrode surface. The electrochemical properties of iodide at the VO(OC₃H₇)₃-PPC film-modified glassy carbon electrode were investigated by cyclic voltammetry, and an anodic peak was observed at approximately +0.71 V (vs. SCE). Based on this, a sensitive and convenient electrochemical method was proposed for the determination of iodide. Flow injection amperometry (FIA) exhibited a good linear relationship with the concentration of iodide in the range of 5 × 10⁻⁷ mol L⁻¹ and 1 × 10⁻³ mol L⁻¹, and the detection limit was 1 × 10⁻⁷ mol L⁻¹. Quantitative recovery of iodide in synthetic samples has been obtained and the interferences from different cations and anions have been studied. The method has been successfully applied to the determination of iodide in dry edible seaweed. The concentrations of iodide measured by this method are in good agreement with those obtained by spectrophotometric method.     

Indirect ultraviolet determination of silver with nickelocyanide ion by flow injection analysis

A flow-injection method for the ultraviolet spectrophotometric determination of silver, based on its reaction with nickelocyanide ion, Ni(CN)²⁻₄, in ammoniacal buffer medium (pH 10) and subsequent measurement of the decrease in the absorption of the Ni(CN)²⁻₄ complex at 275 nm is described. The calibration graph is linear in the range 10–400 μm silver. At a sampling rate of about 60 samples h⁻¹ with 35 μl sample injections, precision was about 1% relative standard deviation. The proposed method was successfully applied to the determination of silver in some common silver minerals.     

Sensitive Flow-Injection Electrochemical Determination of Hydrogen Peroxide at a Palladium Nanoparticle-Modified Pencil Graphite Electrode

A novel flow-injection amperometric method was proposed for the sensitive and enzymeless determination of hydrogen peroxide based on its electrocatalytic reduction at a palladium nanoparticle-modified pretreated pencil graphite electrode in a laboratory-constructed electrochemical flow cell. Cyclic voltammograms of the unmodified and modified electrodes were recorded in pH 7.0 phosphate buffer containing 0.10 M KCl at a scan rate of 50?mV s^?1 for the investigation of electrocatalytic reduction of hydrogen peroxide at the palladium nanoparticle-modified pretreated pencil graphite electrode. Cyclic voltammograms of the pretreated pencil graphite electrode revealed an irreversible oxidation peak and a weak reduction peak of hydrogen peroxide at +1100?mV and –450?mV vs. an Ag/AgCl/KCl saturated reference electrode. However, the reduction of hydrogen peroxide was observed at –100?mV with an increase in current in the cyclic voltammograms of the palladium nanoparticle-modified pretreated pencil graphite electrode compared to the unmodified electrode. These results indicate that the palladium nanoparticle-modified pretreated pencil graphite electrode exhibits efficient electrocatalytic activity for the reduction of hydrogen peroxide. A linear concentration range was obtained between .01 and 10.0?mM hydrogen peroxide with a detection limit of 3.0 µM from flow injection amperometric current–time curves recorded in pH 7.0 phosphate buffer at –100?mV and a 2.0?mL min^?1 flow rate. The novelty of this work relies on its use of a laboratory-constructed flow cell constructed for the pencil graphite electrode using these inexpensive, disposable, and electrochemically reactive modified electrodes for the amperometric determination of hydrogen peroxide in a flow injection analysis system.     

Improving splitless injection with electronic pressure programming

An experimental injection port has been designed for split or splitless sample introduction in capillary gas chromatography; the inlet uses electronic pressure control, in order that the column head pressure may be set from the GC keyboard, and the inlet may be used in the constant flow or constant pressure modes. Alternatively, the column head pressure may be programmed up or down during a GC run in a manner analogous to even temperature programming. Using electronic pressure control, a method was developed which used high column head pressures (high column flow rates) at the time of injection, followed by rapid reduction of the pressure to that required for optimum GC separation. In this way, high flow rates could be used at the time of splitless injection to reduce sample discrimination, while lower flow rates could be used for the separation. Using this method, up to 5 μl of a test sample could be injected in the splitless mode with no discrimination; in another experiment, 2.3 times as much sample was introduced into the column by using electronic pressure programming. Some GC peak broadening was observed in the first experiment.     

Study of the Electrochemical Behavior of Disperse Blue 1‐Modified Graphite Electrodes. Application to the Flow Determination of NADH

The preparation and the electrochemical study of Disperse Blue 1‐chemically modified electrodes (DB1‐CME), as well as their efficiency for the electrocatalytic oxidation of NADH is described. The proposed mediator was immobilized by physical adsorption onto graphite electrodes. The electrochemical behavior of DB1‐CME was studied with cyclic voltammetry. The electrochemical redox reaction of DB1 was found to be reversible, revealing two well‐shaped pair of peaks with formal potentials 152 and ?42 mV, respectively, (vs. Ag/AgCl/3M KCl) at pH 6.5. The current I_p has a linear relationship with the scan rate up to 800 mV s^?1, which is indicative for a fast electron transfer kinetics. The dissociation constants of the immobilized DB1 redox couple were calculated pK₁=4 and pK₂=5. The electrochemical rate constants of the immobilized DB1 were calculated k₁°=18 s^?1 and k₂°=23 s^?1 (Γ=2.36 nmol cm^?2). The modified electrodes were mounted in a flow injection manifold, poised at +150 mV (vs. Ag/AgCl/3M KCl) and a catalytic current due to the oxidation of NADH was measured. The reproducibility was 1.4% RSD (n=11 for 30 μM NADH) The behavior of the sensor towards different reducing compounds was investigated. The sensor exhibited good operational and storage stability.     

流动注射化学发光法测定工业废水中苯酚

采用流动注射技术,研究了高锰酸钾-过氧化氢-苯酚的化学发光行为,对影响化学发光强度的因素进行了试验,建立了流动注射化学发光法测定苯酚的方法。方法的检出限为3×10-5g.L-1,线性范围为1.0×10-4~1.0×10-1g.L,对浓度为4.0×10-4g.L的苯酚作试验,求得其相对标准偏差(n=11)值为1.6%。方法用于废水中苯酚含量的测定,结果与分光光度法测得值一致。