Next generation of flow analysis is based on flow programming.

Concepts of air Segmented Flow Analysis (SFA) and Flow Injection Analysis (FIA) are discussed and compared with the performance of a new method based on comprehensive flow programming. The programmable Flow Injection (pFI), miniaturized on the lab-on-valve platform, can perform tasks that can not be done in any other way, such as Auto calibration by a single standard solution or analysis in a flow-batch mode that exactly simulates the traditional manual batch technique. Performance of pFI is documented by examples of nutrient determinations in sea water.

Programmable flow injection in batch mode: Determination of nutrients in sea water by using a single, salinity independent calibration line, obtained with standards prepared in distilled water.

Interference of the Schlieren effect on sea water analysis by spectrophotometry is caused by the flow of solutions of different ionic strengths through a flow cell. A flow injection assay protocol programmed in a flow-batch format removes this interference and allows the use of a calibration line, obtained in deionized water, for determination of analytes in sea water samples of different salinity. This Single Line Calibration (SLC) technique is validated on the most frequently performed nutrient assays.

The performance of a new linear light path flow cell is compared with a liquid core waveguide and the linear cell is used for spectrophotometric determination of nitrite in sea water at nanomolar concentrations.

The sensitivity of a spectrophotometric assay is enhanced either by increasing the concentration of the target molecules within the flow cell or by extending the length of the light path of the flow cell. Determination of nutrients at nanomolar concentrations in sea water has therefore been based either on the preconcentration of the analyte on a microcolumn from a large volume of sample followed by its elution into a conventional 1-2 cm flow cell, or by the use of Liquid Core Waveguide (LCW) with a light path as long as several meters. In order to evaluate the relative improvements of these different approaches to increasing sensitivity we have developed a preconcentration technique for the determination of nitrite in seawater using the Gries reaction and compare its sensitivity and precision to that of non preconcentration techniques using both LCW and Linear Light Path (LLP) cells of different lengths.

Flow injection programmed to function in batch mode is used to determine molar absorptivity and to investigate the phosphomolybdenum blue method.

The ultimate goal of flow-based analytical techniques is to automate serial assays of a target analyte. However, when developing any reagent-based assay, the underlying chemistry has to be investigated and understood a step, which is almost always the most challenging component of the optimization effort. The difficulty lies in that almost all reagent-based assays were initially developed and optimized in a batch mode, with the aim to perform assays manually, within a time frame of up to 15 min, while flow injection techniques are designed to monitor concentration gradients at times prior to reaching chemical equilibria and while performing up to two assays per minute.

Determination of traces of phosphate in sea water automated by programmable flow injection: Surfactant enhancement of the phosphomolybdenum blue response.

An assay protocol, based on programmable Flow Injection (pFI), is optimized by tailoring flowrates appropriately to the individual steps of an assay, thus allowing sample and reagent metering, mixing, incubation, monitoring and washout to be carried out more efficiently and in different time frames. This novel approach to flow based methods is applied here to optimize the determination of orthophosphate at nanomolar levels. Programmable Flow Injection was also used to facilitate an investigation of the properties of the phosphomolybdenum blue (PMoB) formed during this assay, by using the stop flow technique - an approach that revealed for the first time the influence of surfactants on the kinetics of formation of PMoB and its spectral characteristics.

Programmable Fow Injection. Principle, methodology and application for trace analysis of iron in a sea water matrix.

Automation of reagent based assays by Flow Injection is based on sample processing, in which a sample flows continuously towards and through a detector for monitoring of its components. There are three drawbacks to using this approach. The constant continuous forward flow: continually consumes reagents and generates chemical waste and necessitates a compromise when optimizing the performance of the reagent based assay.

Redesigning flow injection after 40 years of development: Flow programming.

Automation of reagent based assays, by means of Flow Injection (FI), is based on sample processing, in which a sample flows continuously towards and through a detector for quantification of the target analyte. The Achilles heel of this methodology, the legacy of Auto Analyzer®, is continuous reagent consumption, and continuous generation of chemical waste. However, flow programming, assisted by recent advances in precise pumping, combined with the lab-on-valve technique, allows the FI manifold to be designed around a single confluence point through which sample and reagents are sequentially directed by means of a series of flow reversals.

From continuous flow analysis to programmable Flow Injection techniques. A history and tutorial of emerging methodologies.

Automation of reagent based assays, also known as Flow Analysis, is based on sample processing, in which a sample flows towards and through a detector for monitoring of its components. The Achilles heel of this methodology is that the majority of FA techniques use constant continuous forward flow to transport the sample - an approach which continually consumes reagents and generates chemical waste. Therefore the purpose of this report is to highlight recent developments of flow programming that not only save reagents, but also lead by means of advanced sample processing to selective and sensitive assays based on stop flow measurement.

Towards chemiluminescence detection in micro-sequential injection lab-on-valve format: a proof of concept based on the reaction between Fe(II) and luminol in seawater.

Micro-sequential injection lab-on-valve (µSI-LOV) is a well-established analytical platform for absorbance and fluorescence based assays but its applicability to chemiluminescence detection remains largely unexplored. In this work, we describe a novel fluidic protocol and two distinct strategies for photon collection that enable chemiluminescence detection using µSI-LOV for the first time. To illustrate this proof of concept, we selected the reaction between Fe(II) and luminol and developed a preliminary protocol for Fe(II) determinations in acidified seawater.

Determination of dissolved zinc in seawater using micro-Sequential Injection lab-on-valve with fluorescence detection.

This paper introduces the preliminary design and optimization of a micro-Sequential Injection lab-on-valve system (μSI-LOV) with fluorescence detection for the direct determination of trace Zn(2+) in an unacidified seawater matrix. The method capitalizes on the sensitivity and selectivity of FluoZin-3, which was originally designed to measure zinc in living cells. The optimum reaction conditions, sources of blank signal and physical parameters of the μSIA-LOV are evaluated with the requirements of trace metal analysis in mind, namely high sensitivity and low background signals.

Two-parameter monitoring in a lab-on-valve manifold, applied to intracellular H2O2 measurements.

This work introduces, for the first time, simultaneous monitoring of fluorescence and absorbance using Bead Injection in a Lab-on-valve format. The aim of the paper is to show that when the target species, cells immobilized on a stationary phase, are exposed to reagents under well-controlled reaction conditions, dual monitoring yields valuable information. The applicability of this technique is demonstrated by the development of a Bead Injection method for automated measurement of cell density and intracellular hydrogen peroxide.

Sequential injection analysis with dynamic surface tension detection High throughput analysis of the interfacial properties of surface-active samples.

A sequential injection analysis (SIA) system is coupled with dynamic surface tension detection (DSTD) for the purpose of studying the interfacial properties of surface-active samples. DSTD is a novel analyzer based upon a growing drop method, utilizing a pressure sensor measurement of drop pressure. The pressure signal depends on the surface tension properties of sample solution drops that grow and detach at the end of a capillary tip.

Automated capture and on-column detection of biotinylated DNA on a disposable solid support.

This work comprises the development of a technique for the capture of single-stranded DNA on a solid support combined with in situ quantification. The capture is based on the strong and selective interaction between biotinylated DNA and streptavidin-coated agarose beads. Sequential Injection in the lab-on-valve format allows for automated manipulation of all components including the building and disposal of bead columns.

In-situ monitoring of H2O2 degradation by live cells using voltammetric detection in a lab-on-valve system.

This paper describes a method for monitoring the degradation of hydrogen peroxide by cells immobilized on a beaded support. The detection is based on the voltammetric reduction of hydrogen peroxide on a mercury film working electrode, whilst combining the concept of sequential injection (SI) with the lab-on-valve (LOV) manifold allows the measurements to be carried out in real time and automatically, in well-defined conditions. The method is shown to be capable of simultaneously monitoring hydrogen peroxide in the 10-1000 microM range and oxygen in the 160-616 microM range.

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy.

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal.

Immobilization of proteins on agarose beads, monitored in real time by bead injection spectroscopy.

A novel approach to real-time monitoring of protein immobilization resulted in the surprising finding that current immobilization protocols are far from optimized.

Automated method, based on micro-sequential injection, for the study of enzyme kinetics and inhibition.

A micro-reactor system with continual spectrophotometric detection has been operated in Sequential Injection lab-on-valve (SI-LOV) mode and applied to enzyme kinetics and inhibition studies, using acetylcholinesterase (AChE) and angiotensin-converting enzyme (ACE) as model systems. With the advantages of automation, real-time kinetic measurement, and thorough mixing, the SI-LOV micro-reactor system allows for the monitoring of initial reaction rates and determination of reactant concentrations in the reaction mixture, both of which are essential for the determination of kinetic constants for enzymes and inhibitors. Enzyme, substrate, and inhibitor are precisely metered by the syringe pump and delivered to a stirred micro-reactor, followed by a reference scan that establishes the baseline for the following reaction rate measurement.

Atomic absorption spectroscopy for mercury, automated by sequential injection and miniaturized in lab-on-valve system.

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.

Sequential affinity chromatography miniaturized within a "lab-on-valve" system.

An automatically renewable microcolumn, subjected to operation by programmable flow, is presented and for the first time used for separation and quantification of biomolecules on Sepharose Protein A beads, with absorbance measurement at 280 nm and a detection limit of 6 ng mouse IgG microL(-1).

On-line flow cytometry for real-time surgical guidance.

Objective: This study tests the feasibility of using on-line analysis of tissue during surgical resection of brain tumors to provide biologically relevant information in a clinically relevant time frame to augment surgical decision making. For the purposes of establishing feasibility, we used measurement of deoxyribonucleic acid (DNA) content as the end point for analysis.

Methods: We investigated the feasibility of interfacing an ultrasonic aspiration (USA) system with a flow cytometer (FC) capable of analyzing DNA content (DNA-FC).

Accelerated micro-sequential injection in lab-on-valve format, applied to enzymatic assays.

The assay cycle of sequential injection (SI) analysis has been greatly accelerated by simultaneously processing two sample injections within the same manifold. This is achieved through micro-miniaturization of the SI system using the lab-on-valve format (LOV), and by optimizing the assay protocol for stopped-flow reaction rate measurements. The approach has been tested on enzymatic assays of glucose and ethanol, but it is, in principle, applicable to all SI reagent-based assays.

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).

Bead injection ELISA for the determination of antibodies implicated in type 1 diabetes mellitus.

This work introduces a novel analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of type 1 diabetes. There is a clinical need for a rapid and automated assay for GAD65 autoantibodies. Therefore, this method was designed to exploit the advantages of bead injection (BI) analysis for enzyme-linked immunosorbent assays (ELISA).

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.

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.