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MICROFLUIDICS

IntroductionOne of the key objectives for an automated analytical system is to fulfill the needs of

the laboratories that require high-throughput routine analysis of a wide spectrum of

species. The automated analytical system should consist of precise sampling and controlof analytical events that can ensure the assay conditions are well controlled. Highaccuracy and reproducibility over an extended period of time should be achievedcompared to other off-line analysis instruments. The flow injection analysis system (FIA),sequential injection analysis system (SIA), and the most recent μSI-LOV instrumentationshave been proven to meet the requirements of laboratory automation with inherentfeatures of high efficiency, high precision, and a broad spectrum of versatility

FLOW INJECTION ANALYSISThe flow injection analysis (FIA) system performs assay in a format in which the

continuously flowing carrier stream pushes the sample and reagent forward at the

upstream of the system. The reaction product is detected by a detector located at thedownstream of the system. It is an automated technique that allows for easy optimizationand for routine laboratory analysis. Atypical FIA system (Figure 1) consists of a peristalticpump to drive the carrier, a sample injector with bypass channel allowing continuous flowof the carrier solution, a mixing coil, and a detector.

The sample injector in a conventional FIA system employs a two position valve with asample loop with a fixed volume, and a peristaltic pump can only provide a fixed carrier flow rate. Therefore, the injected sample volume and reaction time can not be easilyadjusted by a computer or optimized by software without physically reconfiguring thesystem. The order of the assay events in the FIA system (such as dilution, mixing,separation, etc.) is fixed and strictly bonded to the system hardware configuration.

Operation:

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• Flow rate is one milliliter per minute.• Typical sample volume consumption is 100 microliters per sample.• Typical sampling frequency is two samples per minute.

• FIA assays usually result in sample concentration accuracies of a few percent.

Drawbacks• In certain cases the flow manifolds are complicated, involving multi-channeledsetups that have to be re-configured in order to apply different “chemistries”.• Peristaltic pumps do not provide stable flow on 24-h basis operation and thereforerequire frequent maintenance increasing this way the cost of the analysis.• The continuous flow of reagents even at low flow rates, produces considerableamounts of waste material in the case of 24-h process control applications.

SEQUENTIAL INJECTION ANALYSISThe sequential injection analysis (SIA) system, the second generation FIA system,

was introduced by Ruzicka and Marshall [3] with the aims of improving flexibility, reducingsample and reagent consumptions, and allowing the technique to be more compatiblewith computer automation. A typical SIA system (Figure 2) comprises a bidirectionalsyringe pump, a holding coil, and a multi-position valve(MPV). A detector is one of manyperipherals of the SIA system and can be installed to the desired port on the MPV. Thesyringe pump and the MPV are controlled by a computer with dedicated software. While aconventional FI protocol needs physical reconfiguration of the flow manifold to performdifferent assays, the computer controlled SIA system allows all experimental protocols tobe randomly executed. In a typical assay cycle of the SIA system, the sample and thereagent are injected sequentially through the MPV into the holding coil, forming a well-defined solution profile. This profile is then transported through the mixing coil and into

the detector by the flow reversal action provided by the syringe pump after the MPV hasbeen switched to the detector port. Flow programming, along with random access tosample and reagent ports, provides outstanding versatility since all operating protocolssuch as sample injection, mixing, and separation are controlled precisely by the softwarewithout need for physical reconfiguration of the system.

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Operation• The system is initially filled with a carrier stream .• A zone of sample and a zone of reagent(s) are sequentially aspirated into aholding coil, forming a linear stack.• These zones become overlapped due the parabolic profile induced by differences

between flow velocities of adjacent streamlines.• Flow reversals and flow acceleration further promote mixing.• The multiposition valve is then switched to the detector position, and the flowdirection is reversed, propelling the sample/reagent zones through the flow cell.

Merits• The reagent consumption is minimized due to the discontinuous operation mode.• Less waste is generated compared to FI and in certain cases SI fulfills someprinciples of Green Analytical Chemistry.• There is no need for reconfiguration of the manifold in order to apply different“chemistries”, as all the required steps are carried out through the multi-position valve.

• Syringe pumps offer increased robustness with precise operation and littlemaintenance for process applications.

Drawbacks• A usually reduced sampling rate compared to analogous FI assays.• The difficulty of adaptation of certain FI sub-techniques such as solvent extraction.• The necessity of a suitable software to run the SI system.

BEAD INJECTIONNovel tool for the examination and optimization of protein immobilization protocols, by

measuring the rate and yield of coupling reactions, as they take place on the surface of

agarose beads in a well-stirred microreactor.

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A. System setupB. Flow cell configurationC.Details of flow cell construction

Section B

• Plug ‘‘a’’ focuses the carrier stream into the center of the packed beads.• Plug ‘‘b’’ helps retain the beads within the optical path.

Section C• Optical fibers hold plug ‘‘a’’ in place.• Teflon tubing in the waste channel is cut at an angle to hold plug ‘‘b’’ in place andto prevent clogging

Application of BI• The methodology BI is a useful tool for quality control of agarose-basedchromatographic supports, as well as for the optimization of a wide variety of

immobilization chemistries, as used for synthesis of chromatographic supports,immobilization of enzymes, and derivatization of biosensing surfaces.

OperationBeadinjection7 uses suspended beads as carriers for reactive groups or reagents. Thebead suspension is injected into the flowchannel, where the beads are trappedand perfused by analyte solutions, buffers and/or auxiliary reagents.(Bio)chemical reactions taking place at the bead surfaces can thus be monitoredin real time, either directly on the solid phase or by monitoring the eluting liquidphase. In this work, the monitoring

of beads in the jet ring cell configuration was performed by absorbancemeasurement or by fluorescence

Schematic of SI in Chromatographic Seperation

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Applications• Pharmaceutical analysis and quality control.The application of automated FIA procedures in the quality control of pharmaceuticals and in pharmaceutical research

A) Pharmacopoeial assaysThe regulations concerning quality control of pharmaceutical formulations(especially solid dosage forms) require to carry out content uniformity tests withlarge sets of individual tablets and to examine the liberation of the activesubstance from the formulation (dissolution tests for tablets or ointments). Thesetasks may be solved by anautomated FIA with its excellent reagent economy and capacity of samplingfrequencies close to 100 h-1. Application of multi-sensing devices (UVVIS diode-array detectors, FT-IR detection, multichannel electrochemical detection withelectrode arrays and combination of two or more serial or parallel detectors in a

single FIA manifold) offerspossible solution of the selectivity problems that may occur when analysingmulticomponent formulations.B) Pharmaceutical research and industry FIA with its capability of automated analyticaldata collection represents a method of choice in drug discovery activities(screening of potential drugs, namely in vitro/in vivo monitoring of their biologicaleffects reflected in specific chemical changes of examined systems in real time)in pharmacologicaldrug testing (in addition to conventional pharmacokinetic and drug-proteinbinding studies also examination of responses of cells, tissues, isolated organs

and biological membranes to the effect of drugs) and in pharmaceuticaltechnology(e.g., optimisation and monitoring of technological processes in drug productionincluding biotransformations in biotechnology).• Food analysis.• Environmental monitoring.• Bioanalytical applications.Pre-existing sequential injection methods can be quickly adapted onto a Lab-on-Valve (LOV) manifold to develop microassays that substantially reduced reagentconsumption and waste generation.1.Affinity separationsBead Injection Electrospray Mass Spectrometry (BIEMS) has been introduced asa new method4 for automated affinity purification combined with on-line analysisof an eluted conjugate. Strong binding between biotin and streptavidin serves asthe basis where avidin coated Sepharose beads is used tocapture a biotin derivative. The LOV manifold automates the assemble of a freshbead column which is used to capture it biotin conjugate. The method achievesfast capture-purification-release for MS analysis using only a few microliters of sample. This has been demonstrated for assays of the enzyme β-galactosidase

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in human cell lysates. The overall goal of this work is to use the BIEMS system toscreen newborn baby’s blood for defective enzymatic activities, which areindicative of the presence of genetic diseases. In contrast to batch enzymaticassays that require several hours for the capture-release of a conjugate, theBIEMS using continuous flow achieves capture and elution in under five minutes.

2. Fermentation MonitoringThe LOV system has been used to monitor aerobic fermentations3 of E. Coli andS. Cerevisiae cell cultures. Due to its compact size, the analyzer was situated on-line and used colorimetric methods to monitor important nutrient levels in thefermentation broth. Stopped-flow SI mode was used to produce linear calibrations of ammonia (3-1200 ppm), glucose (35-1000 ppm), glycerol (20-120ppm) and free iron (80-400 ppm).Sampling was performed at five-minute intervals over the course of several days.The assays only required a few microliters of sample, which is of particular importance to a small-scale experimental fermentation. Moreover, the large boresize found in the LOV prevented clogging for fermentation debris making

this μSI system very reliable.3. Functional cellular assaysThe identification and characterization of drug candidates require that theefficacy of a drug be determined. Such assays are referred to as functionalassays, and are designed to classify a drug as an agonist or antagonist depending on whether a biological response is invoked or inhibitedThe LOV coupled to a fluorescence microscope for detection has provedto be an ideal tool for automating these assays.. Dose response curvesconstructed from the bead injection assay can discriminate a partial agonistresponse from a full agonist response allowing these drugs to be rated for pharmacological purposes.

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