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.     

Determination of total ammonium-nitrogen and free ammonia in a fermentation medium by sequential injection analysis

Summary Automated methods for the determination of ammonium and ammonia are reviewed, and techniques based on gas diffusion using a semi-permeable membrane were selected for the determination of both total ammonium-nitrogen and free ammonia in fermentation samples. A simple and robust instrument based on sequential injection analysis (SIA) consisting of a piston pump and two selector valves was used. Two different methods of ammonia detection, the Berthelot method and detection using pH-indicators, have been evaluated and compared. The indicator method showed higher reproducibility and the range of determination could easily be adjusted to match the concentrations of the samples. The range of determination could be adjusted from 0.05 mmol/l to 350 mmol/l, depending on choice of acceptor solution. This method has been evaluated with fermentation medium samples and tested on-line in a yeast fermentation process.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve system

Monitoring extracellular acidification rates (ECARs) is important for the study of cellular activities, since it allows for the evaluation of factors that alter metabolic function, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. While the light addressable potentiometric sensor (Cytosensor Microphysiometer) has been the principal tool for ECARs measurement in the past, this work introduces a novel method that exploits an immobilized pH indicator on the surface of microcarrier beads (Sephadex) and is probed with a fiber optic coupled spectrophotometer. Likewise, live cells under investigation were also immobilized on microcarrier beads (Cytopore). These beads are metered, transported and monitored within a microfluidic system, termed as the Lab-on-Valve (LOV). Use of carrier beads in conjunction with Bead Injection Spectrophotometry and a Lab-on-Valve module (BIS-LOV), makes ECAR measurements reliable and automated. The feasibility of the BIS-LOV approach is demonstrated measuring ECARs of the mouse hepatocyte cell line, TABX.2S, grown on Cytopore beads packed within the central channel of the LOV system. These immobilized cells were perfused in a phosphate buffer carrier solution (capacity: 1 mmol L(-1), pH 7.4). Protons extruded from 10(5) to 10(6) cells were accumulated during a stopped flow period of 220 s followed by a pH measurement, detected by changes in absorbance of the pH indicator bonded to the microcarrier beads. Addition of metabolic inhibitors (sodium azide, oxamic acid) to the carrier buffer solution can induced an increase or decrease of the basal proton extrusion rate in a very reproducible manner. Comparison of the BIS-LOV technique to the Cytosensor microphysiometer and literature confirms the validity of this novel approach, highlighting its advantages and suggesting future improvements that will make the BIS-LOV a practical tool for routine ECARs measurement.     

Real-time monitoring of lactate extrusion and glucose consumption of cultured cells using a lab-on-valve system

Microsequential injection (microST) provides microfluidic operations that are ideally suited for cellular function studies and as a means of validating targets for drug discovery. MicroSI carried out within the lab-on-valve (LOV) manifold, is an ideal platform for spectroscopic studies on living cells that are grown on microcarrier beads and kept thermostated while their metabolism is probed in real-time. In this paper a microbioreactor is integrated into the LOV manifold allowing measurement of cellular lactate extrusion and glucose consumption rates of a cell culture that is automatically renewed prior to each measurement. Glucose consumption and lactate extrusion are monitored using NAD-linked enzymatic assays. The microSI-LOV setup has demonstrated a linear analysis range of 0.05-1.00 mM for lactate and 0.1-5.6 mM for glucose. These assays were conducted in a serial fashion requiring 3 microL of cellular perfusate and 10 s for glucose determination and 30 s for the lactate assay. Overall waste generated per lactate/glucose assay is < 200 microL. This work was performed using two different transfected hepatocyte cell lines, which adhere to Cytopore microcarrier beads. This novel approach to metabolic screening allows for the rapid evaluation of the effects of dosing cells with chemical agents.     

Micro solid phase spectrophotometry in a sequential injection lab-on-valve platform for cadmium,zinc, and copper determination in freshwaters

This work describes the development of a solid phase spectrophotometry method in a μSI-LOV system for cadmium, zinc, and copper determination in freshwaters. NTA (Nitrilotriacetic acid) beads with 60–160 μm diameter were packed in the flow cell of the LOV for a μSPE column of 1 cm length. The spectrophotometric determination is based on the colourimetric reaction between dithizone and the target metals, previously retained on NTA resin. The absorbance of the coloured product formed is measured, at 550 nm, on the surface of the NTA resin beads in a solid phase spectrophotometry approach.     

On-line monitoring of glucose and penicillin by sequential injection analysis

A sequential injection analysis (SIA) system has been developed for on-line monitoring of glucose and penicillin during cultivations of the filamentous fungus Penicillium chrysogenum. The SIA system consists of a peristaltic pump, an injection valve, two piston pumps, two multi-position valves and a detector. The glucose analyzer is based on an enzymatic reaction using glucose oxidase, which converts glucose to glucono-lactone with formation of hydrogen peroxide and subsequent detection of H₂O₂ by a chemiluminescence reaction involving luminol. The penicillin analysis is based on formation of penicilloic acid by penicillinase. Penicilloic acid is detected either by a chemiluminescence method or by a decolorization method. In the chemiluminescence method the penicilloic acid is quantified by its quenching effect on the chemiluminescence signal obtained when luminol reacts with iodine. In the decolorization method the penicilloic acid is detected spectrophotometrically by the decrease in the absorbance of an iodine-starch complex.     

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.     

Sequential injection lab-on-valve system for the on-line monitoring of hydrogen peroxide in lens care solutions

A sequential injection lab-on valve (SI-LOV) method for the enzymatic determination of hydrogen peroxide was developed. The spectrophotometric assay is based on the reaction between hydrogen peroxide and ABTS (2,2′-Azino-bis(3-Ethylbenzothiazoline 6-sulfonic acid)) in the presence of the enzyme HRP (horseradish peroxidase). The produced oxidized ABTS is measured at 410 nm. The sample consumption was 15 µL/assay and the consumption of HRP and ABTS was 34.6 mg L^{− 1} and 0.06 g L^{− 1}, respectively with a determination rate of 45 h^{− 1}.Relative deviations lower than 9.0% were found when the results were compared to those obtained by the reference procedure in the analysis of bleaches and disinfection solutions for contact lenses. With the incorporation of an in-line dilution (dialysis) process, was possible to attain a response range up to 342 mg L^{− 1} of hydrogen peroxide. The developed method was applied to monitor on-line of the disinfection–neutralization process of contact lenses. The study of two different one-step systems for cleaning contact lenses demonstrated that the neutralization of the hydrogen peroxide is completed within 6 h as recommended by the manufactures. The developed flow method was proved to be a useful tool for monitoring the dynamic process of disinfection–neutralization.     

An electrochemical assay for the determination of Se (IV) in a sequential injection lab-on-valve system

A sequential injection lab-on-valve (LOV) unit, integrating a miniaturized electrochemical flow cell (EFC), has been constructed for the determination of trace amounts of Se (IV) by employing cathodic stripping voltammetry (CSV) technique. The procedure is carried out on a mercury film coated glassy carbon electrode. The analyte solution and electrolyte solution were continuously aspirated and merged in the holding coil (HC) by using a single syringe pump, which were afterwards pushed into the EFC, where the peak current was generated during the subsequent deposition/stripping procedure and measured as the basis of quantification. Assay parameters were optimized in order to achieve the best analytical performance, including mercury film preparation, supporting electrolyte composition, deposition potential and deposition time, and flow variables in the LOV. By loading a sample volume of 500 μL, a linear calibration graph was derived within 1-600 μg L⁻¹, and a detection limit (3б) of 0.11 μg L⁻¹ was achieved along with a sampling frequency of 20 h⁻¹. By integrating the EFC into the LOV unit, the assembling system not only minimized the sample/reagent consumption and waste generation, but also enhanced the sampling frequency. The work itself extended the applications of electrochemical detection techniques and provided a good platform for Se (IV) electrochemical analysis.     

Automated solid-phase extraction hyphenated to voltammetry for the determination of quercetin using magnetic nanoparticles and sequential injection lab-on-valve approach

In this study, an automated sequential injection lab-on-valve (SI-LOV) system was designed for the on-line matrix removal and preconcentration of quercetin. Octadecyl functionalized magnetic silica nanoparticles were prepared and packed into the microcolumn of the LOV as adsorbents. After being adsorbed through hydrophobic interaction, the analyte was eluted and subsequently introduced into the electrochemical flow cell by voltammetric quantification. The main parameters affecting the performance of solid-phase extraction, such as sample pH and flow rate, eluent solution and volume, accumulation potential and accumulation time were investigated in detail. Under the optimum experimental conditions, a linear calibration curve was obtained in the range of 1.0 × 10(-8) to 1 × 10(-5) mol L(-1) with R(2) = 0.9979. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.3 × 10(-9) and 4.3 × 10(-9) mol L(-1), respectively. The relative standard deviation (RSD) for the determination of 1.0 × 10(-6) mol L(-1) quercetin was found to be 2.9% (n = 11) along with a sampling frequency of 40 h(-1). The applicability and reliability of the automated method described here had been applied to the determination of quercetin in human urine and red wine samples through recovery experiments, and the obtained results were in good agreement with those obtained by the HPLC method.     

Novel approach for mono-segmented flow micro-titration with sequential injection using a lab-on-valve system: a model study for the assay of acidity in fruit juices

A new concept for micro-titration using a "lab-on-valve"(LOV) system with sequential injection of mono-segmented flow is proposed. The performance of the system was demonstrated by the assay of acidity in fruit juices which is based on acid-base neutralization. A standard/sample solution containing citric acid, indicator, sodium hydroxide, were sandwiched between air segments and were aspirated in microliter volumes through a selection valve into a holding coil. The acid, indicator, and base were mixed by flow reversal. After removing air segments, the solution was pushed to the detector for monitoring of the change in absorbance of the indicator color, which depended on the concentration of the remaining base. With LOV, microliter volumes of the solution can be detected without dispersion of the color zone. A calibration graph (plot of absorbance vs. acidity value) in the range of 0.2-1.2% (w/v) as citric acid was established. Sample throughput of 30 sample h(-1) and good reproducibility (RSD = 1.2%, n= 11 for 0.6% acidity) were achieved. The procedure has been applied to determine acidity in fruit juices.     

Label dilution method: a novel tool for bioligand interaction studies using bead injection in the lab-on-valve format

This work introduces a novel method, label dilution, which is analogous to the well-established isotope dilution method. The principle is tested on a model system of commercially available antibodies and protein-coated Sepharose beads and implemented using micro-bead injection in the lab-on-valve format. This micro-scale method uses a labeled form of the target molecule as an internal standard. Label dilution employs ratiometric measurements using the absorbance signals from the label and the target molecules for quantitative determination of an analyte. The label dilution method is shown to discriminate between selective and non-selective binding and provides a means for monitoring bioligand interactions in real time. With a detection limit of 470 ng of IgG, this method provides a sensitive, automated technique for the determination of low-level analytes in complex samples. This technique has been developed with the aim of using it to facilitate diabetes research in which the interactions between autoantibodies and the molecules they target are a central focus.     

Sequential injection lab-on-valve simultaneous spectrophotometric determination of trace amounts of copper and iron

A sequential injection (SI) method in a lab-on-valve (LOV) format for simultaneous spectrophotometric determination of copper and iron has been devised. The detection chemistry is based on the complex formation of 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]aniline (5-Br-PSAA) with copper(II) and/or iron(II) at pH 4.6. Copper(II) reacts with 5-Br-PSAA to form the complex which has an absorption maximum at 580 nm but iron(III) does not react. In the presence of a reducing agent only iron(II)-5-Br-PSAA complex is formed and detected at 558 nm. Under the optimum experimental conditions, the determinable ranges are 0.1-2 mg l⁻¹ for copper and 0.1-5 mg l⁻¹ for iron, respectively, with a sampling rate of 18 h⁻¹. The limits of detection are 50 μg l⁻¹ for copper and 25 μg l⁻¹ for iron. The relative standard deviations (n = 15) are 2% for 0.5 mg l⁻¹ copper and 1.8% for 0.5 mg l⁻¹ iron when determined in standard solutions. The recoveries range between 96 and 105% when determining 0.25-2 mg l⁻¹ of copper and 0.2-5 mg l⁻¹ of iron in artificial mixtures at copper/iron ratios of 1:10 to 5:1. The proposed SI-LOV method is successfully applied to the simultaneous determination of copper and iron in multi-element standard solution and in industrial wastewater samples.     

Determination of cadmium with a sequential injection lab-on-valve by anodic stripping voltammetry using a nafion coated bismuth film electrode

This work exploited a sequential injection lab-on-valve (LOV) system for the determination of cadmium by anodic stripping voltammetry (ASV). A miniaturized electrochemical flow cell (EFC) was fabricated in LOV, in which a nafion coated bismuth film electrode was used as working electrode. The cadmium was electrodeposited on the electrode surface in bismuth solution, and measured with the subsequential stripping scan. Under optimal conditions, the proposed system responded linearly to cadmium concentrations in a range 2.0-100.0 μg L⁻¹. The detection limit of this method was found to be 0.88 μg L⁻¹. By loading a sample volume of 800 μL, a sampling frequency of 22 determinations h⁻¹ was achieved. The repeatability expressed as relative standard derivation (R.S.D.) was 3.65% for 20 μg L⁻¹ cadmium (n = 11). The established method was applied to analysis of trace cadmium in environmental water samples and the spiked recoveries were satisfactory.     

Extraction and preconcentration of trace levels of cobalt using functionalized magnetic nanoparticles in a sequential injection lab-on-valve system with detection by electrothermal atomic absorption spectrometry

A new approach to performing extraction and preconcentration employing functionalized magnetic nanoparticles for the determination of trace metals is presented. Alumina-coated iron oxide nanoparticles were synthesized and used as the solid support. The nanoparticles were functionalized with sodium dodecyl sulfate and used as adsorbents for solid phase extraction of the analyte. Extraction, elution, and detection procedures were performed sequentially in the sequential injection lab-on-valve (SI-LOV) system followed by electrothermal atomic absorption spectrometry (ETAAS). Mixtures of hydrophobic analytes were successfully extracted from solution using the synthesized magnetic adsorbents. The potential use of the established scheme was demonstrated by taking cobalt as a model analyte. Under the optimal conditions, the calibration curve showed an excellent linearity in the concentration range of 0.01–5 μg L^?1, and the relative standard deviation was 2.8% at the 0.5 μg L^?1 level (n = 11). The limit of detection was 6 ng L^?1 with a sampling frequency of 18 h^?1. The present method has been successfully applied to cobalt determination in water samples and two certified reference materials.     

Successive determination of urinary protein and glucose using spectrophotometric sequential injection method

A new sequential injection (SI) system with spectrophotometric detections has been developed for successive determination of protein and glucose. The protein assay is based on ion-association of protein with tetrabromophenolphthalein ethyl ester (TBPE) in the presence of Triton X-100 at pH 3.2. The blue product is monitored for absorbance at 607 nm. For glucose, hydrogen peroxide, generated by the oxidation of glucose in the presence of glucose oxidase immobilized on glass beads packed in a minicolumn, is monitored using iron-catalyzed oxidation reaction of p-anisidine to form a red colored product (520 nm). The SI procedure takes advantage in performing the protein assay during the incubation period for glucose oxidation. Linear ranges were up to 10 mg dL⁻¹ human serum albumin (HSA) with a limit of detection (LOD) (3σ) of 0.3 mg dL⁻¹, and up to 12.5 mg dL⁻¹ glucose with LOD of 0.08 mg dL⁻¹. R.S.D.s (n = 11) were 2.7% and 2.5% (for 1 mg dL⁻¹ and 5 mg dL⁻¹ HSA) and 1.4% (9 mg dL⁻¹ glucose). Sample throughput for the whole assay of both protein and glucose is 6 h⁻¹. The automated system has been demonstrated for the successive assay of protein and glucose in urine samples taken from diabetic disease patients, with good agreement with the other methods. This developed SI system is an alternative automation for screening for diabetic diagnosis.     

Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor

This paper describes a microquantitative method for glucose determination in situ of living cells in real-time. In this novel technique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injection lab-on-valve system (microSI-LOV). Glucose sensing is performed through the use of a two-step, NAD-linked enzymatic process. The course of the assay is monitored in real-time, by absorbance of NADH at 340 nm. The microsequential assay based on plug/nozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM. The design of the (microSI-LOV) system allows the assay to be carried out using only 40 microL of the enzyme reagent and 3 microL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopore beads. Rapid cellular glucose consumption, in this technique, is facilitated by a high cell density, which allows a large number of cells (10(4)-10(5)) to be retained in a very small volume (3 microL). In turn, this cell density results in the rapid depletion of glucose from the cell medium over short time periods (< 2 min). In conjunction with the assay development, the plug/nozzle design and its ramifications on mixing in general are presented and discussed.     

A double-line sequential injection system for the spectrophotometric determination of copper, iron, manganese, and zinc in waters

A double-line sequential injection system was developed for the spectrophotometric determination of several metal ions in waters. The proposed double-line configuration was used to enable adding sample and chromogenic reagents as merging zones. The methodology was applied to the spectrophotometric determination of copper, iron, manganese, and zinc in samples of diverse origins at the range of 0.15-5.00, 0.10-10.0, 0.48-4.00, and 0.11-5.00 mg/L, respectively. Different chromogenic reagents and detection wavelengths were used. The chromogenic reagents for iron and manganese were 1,10-phenanthroline and formaldoxime, respectively. Copper and zinc were both determined using the analytical reagent zincon. Analytical characteristics of the methodology, such as manifold parameters, buffer pH, and reagent concentrations were optimized, and interference of some of the metal ions commonly present in water sample was assessed. Results of the analysis were in agreement with those obtained by atomic absorption spectrometry. Repeatability, expressed as the relative standard deviation for 10 consecutive injections of water samples, was lower than 6%. The determination rate was approximately 36/h.     

Gas diffusion sequential injection system for the spectrophotometric determination of free chlorine with o-dianisidine

A gas diffusion sequential injection system for spectrophotometric determination of free chlorine is described. The detection is based in the colorimetric reaction between free chlorine and a low toxicity reagent o-dianisidine. A gas diffusion unit is used to isolate free chlorine from the sample in order to avoid possible interferences. This feature results from the conversion of free chlorine to molecular chlorine (gaseous) with sample acidification. With minor changes in the operating conditions, two different dynamic ranges were obtained enhancing the application both to water samples and bleaches. The results obtained with the developed system were compared to the reference method, iodometric titration and proved not to be statistically different. A detection limit of 0.6 mg ClO⁻/L was achieved. Repeatability was evaluated from 10 consecutive determinations being the results better than 2%. The two dynamic ranges presented different determination rates: 15 h⁻¹ for 0.6-4.8 mg ClO⁻/L (water samples) and 30 h⁻¹ for 0.047-0.188 g ClO⁻/L (bleaches).     

On-line monitoring of phosphate in natural water and effluent streams using sequential injection analysis

Automation of the molybdenum blue method by sequential injection (SIA) for the on-line monitoring of phosphate in natural waters is presented. Although sequential injection analysis runs at one fifth the speed of conventional FIA, it presents many advantages such as simplicity of the manifold, robustness and computer compatibility. A reduction in reagent and sample consumption is also observed. Flow detector fouling does not occur in SIA manifolds as the detector is in contact with water between analysis. The proposed SIA analyser is able to monitor phosphate in the range 0–70 mg l^–1 with a standard deviation of 0.9%. The detection limit is 0.5 mg l^–1 PO ₄ ^3– .