WO2001076743A2 - Appareil et procede destines a l'analyse d'un analyte fluidique - Google Patents

Appareil et procede destines a l'analyse d'un analyte fluidique Download PDF

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Publication number
WO2001076743A2
WO2001076743A2 PCT/GB2001/001650 GB0101650W WO0176743A2 WO 2001076743 A2 WO2001076743 A2 WO 2001076743A2 GB 0101650 W GB0101650 W GB 0101650W WO 0176743 A2 WO0176743 A2 WO 0176743A2
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Prior art keywords
sample
chamber
flow rate
analytical device
detection means
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PCT/GB2001/001650
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English (en)
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WO2001076743A3 (fr
Inventor
Elizabeth Anne Howlett Hall
Ian Stuart Harding
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Cambridge University Technical Services Limited
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Application filed by Cambridge University Technical Services Limited filed Critical Cambridge University Technical Services Limited
Priority to AU2001248542A priority Critical patent/AU2001248542A1/en
Publication of WO2001076743A2 publication Critical patent/WO2001076743A2/fr
Publication of WO2001076743A3 publication Critical patent/WO2001076743A3/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01F—MEASURING VOLUME, VOLUME FLOW, MASS FLOW OR LIQUID LEVEL; METERING BY VOLUME
    • G01F1/00—Measuring the volume flow or mass flow of fluid or fluent solid material wherein the fluid passes through a meter in a continuous flow
    • G01F1/704—Measuring the volume flow or mass flow of fluid or fluent solid material wherein the fluid passes through a meter in a continuous flow using marked regions or existing inhomogeneities within the fluid stream, e.g. statistically occurring variations in a fluid parameter
    • G01F1/708—Measuring the time taken to traverse a fixed distance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • G01N35/085—Flow Injection Analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0457—Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties

Definitions

  • the present invention relates to the field of analysis systems able to measure the flow rate or viscosity of a fluid sample.
  • the flow rate or viscosity is used in calculating the concentration of an analyte in a fluid sample.
  • the invention relates in particular to the design of a flow channel and sample chamber attached thereto for use as or in an analytical measurement system.
  • ⁇ TAS Miniaturized total analysis systems
  • ⁇ TAS miniaturized total analysis systems
  • FIA flow injection analysis
  • detection methodology as diverse as electrochemistry and optics can be incorporated.
  • Miniaturized total analysis systems ⁇ TAS
  • ⁇ TAS miniaturized total analysis systems
  • These include the cheapness and replaceability of components which can be fabricated in large-volume batches, the low operational costs associated with low consumption of chemicals, analytes and power 1 ", and the promise of a 'black box' unit which will give reliable analyses in the hands of unskilled operators.
  • Another advantage of such a miniaturized system is its potential to be made into a portable device.
  • Miniature liquid-moving systems have been formed using micropumps ,v v ' v ' or osmotic pumping" 1 '"" but it is quite common just to use a separate pumping system.
  • all these methods compromise the advantages of the ⁇ TAS approach, either by increasing the fabrication complexity and hence cost or by reducing the portability of the final sensor.
  • Capillary-fill is a known means of achieving sample and/or reagent delivery; this can achieve integrated sample collecting and measurement and consists of a device possessing a cavity or cavities having dimensions small enough to enable sample liquid to be drawn into the cavity by capillary action.
  • /ij m is the diffusion-limiting current which is measured and which is proportional to c m the bulk concentration of the electroactive analyte species.
  • D the diffusion coefficient should be constants.
  • W the electrode width, x e the electrode length, h the cell half height, and of the channel width are constants which describe the channel and Vf is the volume flow rate through the channel.
  • V s must be known, but without a pumping system to control flow, this varies according to sample head-height in the reservoir above the channel and according to sample to sample variation (eg changes in viscosity). The 'fill and flow' channel has thus gained little favour in the design of analytical systems.
  • a first aim of the present invention is to provide an assay for the viscosity of a fluid sample or its flow rate through a sensor.
  • the assay should ideally be carried out with minimal user intervention and better reproducibility, reliability and tolerance of sample variation than is found in the prior art.
  • a second aim is to provide an assay for the concentration of an analyte in a fluid sample.
  • This assay should also ideally be carried out with minimal user intervention and better reproducibility, reliability and tolerance of sample variation than is found in the prior art.
  • the measurement of fluid flow rate/viscosity is used to achieve this second aim.
  • a further aim is to provide said assays without requiring pressurising or pumping technology and avoiding the cost and complexity of automatic liquid dispensing systems.
  • an analytical device for measuring the flow rate or viscosity of a fluid sample
  • the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to flow of the sample, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location in the chamber and the flow rate dependent detection means is used to establish when the sample is at said location.
  • the analytical device further comprises a channel, wherein the chamber allows the sample to flow through the channel and the flow rate dependent detection means is sensitive to the rate of sample flow through the channel.
  • the chamber may be vertical.
  • the feature which causes a change in sample flow rate when the sample is at a location is a change in the cross-sectional area of the chamber.
  • the change in cross-sectional area is a discontinuity in cross-sectional area.
  • the chamber has two or more changes in cross- sectional area.
  • the analytical device is adapted to calculate the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one feature to a second feature.
  • the detection means may comprise a sensor for a component of the sample.
  • the detection means may comprise a sensor for a component added to the sample.
  • the detection means may comprise a sensor for matter produced responsive to a component of the sample.
  • the detection means comprise an electrochemical sensor and/or an optical detection means.
  • the analytical device has a flow-rate restriction means.
  • the flow rate restriction means is a porous plug.
  • the flow rate restriction means may comprise an electrochemical sensor.
  • the analytical device has a waste collection means.
  • the waste collection means may comprise a waste guide means and a waste collector.
  • the waste guide means is preferably a rod.
  • an analytical device for measuring the concentration of an analyte in a fluid sample
  • the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to the rate of fluid flow, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location and the detection means is used to establish when the sample is at said location and thereby to calculate the rate of sample flow through the chamber at a given point in time.
  • the analytical device further comprises a channel, wherein fluid flows from the chamber through the channel and the flow rate dependent detection means is sensitive to the rate of fluid flow through the channel.
  • the flow rate dependent detection means may comprise a sensor for the analyte.
  • the analytical device may have a reaction zone which functions to produce chemical species in a concentration which depends on the concentration of the analyte.
  • the reaction zone may comprise means to convert the analyte to a chemical species which can be detected by a chemical species sensor.
  • the reaction zone may displace or produce detectable matter in response to the presence of the analyte.
  • the reaction zone may remove chemical species from a sample in response to the presence of analyte.
  • the reaction zone may comprise a layer of reaction component on a channel wall.
  • the reaction zone may comprise reaction component immobilised on beads in the channel or chamber.
  • the amount of chemical species converted, produced or displaced from the reaction zone may be independent of flow rate.
  • the reaction zone may be selected such that its functionality is altered by the concentration of analyte.
  • the reaction zone may comprise an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte.
  • the analyte may be any chemical which has an effect on the reaction zone.
  • the reaction zone may comprise antibody to an analyte, wherein the reaction zone carries out immunoassays including sandwich and/or competitive i munoassays.
  • the detection means may comprise and electrochemical and/or optical detection means.
  • the sample flows through under the influence of gravity. More preferably, the chamber is vertical.
  • the feature which causes a change in sample flow rate when the sample is at a known location may be a change in the cross-sectional area of the chamber.
  • the chamber comprises at least two changes in cross-sectional area.
  • the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change.
  • the change in cross-sectional area is a step change.
  • the analytical device has a flow-rate restriction means.
  • the flow rate restriction means is a porous plug.
  • the flow rate restriction means may be an electrochemical sensor.
  • the analytical device preferably has a waste collection means. More preferably, the waste collection means comprises a waste guide means and a waste collector. Most preferably, the waste guide means is a rod.
  • the analytical device may comprise a plurality of chambers. A or each chamber may have a plurality of detection means for different analytes.
  • a method for measuring the flow rate or viscosity of a fluid sample comprising the steps of:
  • the fluid sample flows from the chamber into a channel and the flow rate dependent detection means is sensitive to the flow rate of the sample through the channel. More preferably, the sample flows under the influence of gravity. Most preferably, the chamber is vertical.
  • the change in cross-sectional area is a step change in the cross-sectional area of the chamber.
  • the chamber may comprise at least two changes in cross-sectional area.
  • the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change.
  • the detection means may comprise a sensor for a component of the sample.
  • the detection means may comprise a sensor for a component added to the sample.
  • the detection means may comprise a sensor for matter produced responsive to a component of the sample.
  • the detection means may comprises an electrochemical and/or optical detection means.
  • the channel has a flow-rate restriction means. More preferably, the flow rate restriction means is a porous plug.
  • the fluid preferably flows into a waste collection means.
  • the waste collection means may comprise a waste guide means and a waste collector.
  • the waste guide means preferably comprises a rod.
  • a fourth aspect of the present invention there is provided a method for measuring the concentration of an analyte in a fluid sample, comprising the steps of:
  • fluid flows from the chamber into a channel, wherein the flow rate dependent detection means is sensitive to the rate of fluid flow within the channel.
  • the flow rate dependent detection means may comprise a sensor for the analyte.
  • the analyte may be converted to a chemical species which can be detected by the a sensor for said chemical species.
  • the method may further comprise the step of causing detectable matter to enter or leave the fluid sample in a concentration which depends on the concentration of the analyte.
  • a chemical species may be displaced or produced in response to the presence of the analyte.
  • Detectable matter may enter or leave the fluid sample in a concentration which depends on the concentration of the analyte as a result of the action of a layer of reaction component on a channel wall within the channel or chamber.
  • Detectable matter may enter or leave the fluid sample in a concentration which depends on the concentration of the analyte as the result of the action of a component immobilised on a support within the channel or chamber.
  • the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be independent of flow rate.
  • the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be responsive to the concentration of analyte.
  • the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be controlled by an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte.
  • the analyte may be any chemical which causes detectable matter to enter or leave the fluid sample.
  • the method may further comprise the step of carrying out immunoassays, including sandwich or competitive immunoassays, using an antibody to the analyte.
  • the detection means may comprises an electrochemical and/or optical detection means.
  • the sample flows under the influence of gravity. More preferably, the chamber is vertical.
  • the change in cross-sectional area may be a step change in cross-sectional area.
  • the chamber may have two or more changes in cross-sectional area.
  • the method may further comprise the step of calculating the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one change to a second change.
  • the channel has a flow-rate restriction means. More preferably, the flow rate restriction means is a porous plug.
  • the flow rate restriction means may comprise an electrochemical sensor.
  • the fluid sample flows into a waste collection means.
  • the waste collection means comprises a waste guide means and a waste collector.
  • the waste guide means is a rod.
  • the method may further comprise the step of using multiple chambers. More than one detection means may be used for different analytes per sample.
  • a chamber for holding a fluid sample and allow said fluid sample to drain under the influence of gravity comprising at least one change in cross-sectional area.
  • the change in cross-sectional area is a step change in cross-sectional area.
  • the chamber comprises at least two changes in cross-sectional area.
  • a computer program for determining the flow rate or viscosity of a fluid sample inserted into and flowing from a chamber, the computer program comprising program instructions for a) analysing a time course of readings from a flow rate dependent detection means to determine when a fluid sample passed one or more features in the cross-section of the chamber;
  • the features are step changes in the cross-sectional area of the chamber.
  • the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features.
  • a computer program for determining the concentration of an analyte in a fluid sample inserted into and flowing from a chamber comprising program instructions for
  • the features are step changes in the cross-sectional area of the chamber.
  • the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features.
  • Figure 1 A and IB show a schematic illustration of an alternative apparatus including 'sample retention' in the channel design, which can be used for single assay.
  • Figure 1 A shows a side view
  • Figure IB shows a top view;
  • FIG 2 is similar to Figure 1 except the Channel contains an electrochemical detection system.
  • the electrodes are in contact with the fluid passing through the channel;
  • Figure 3 is a schematic illustration of one embodiment of the present invention which is similar to figure 2 except it includes 'sample output and collection' which can be used in a multiple sample assay system;
  • Figure 4 shows embodiments of Figure 3 where overall flow reduction is include by A placing a porous plug between the Chamber-reservoir and the Channel; B placing a porous plug in the outlet port; C including a rod or tube annulus to constrict the cross section of the entry to the Channel. This tube may also be used to introduce reagents or wash;
  • Figure 5 illustrates some combinations of application of the Chamber together with reagents
  • Figure 6 illustrates a sensing area(s) within the Channel
  • Figure 7 illustrates the procedure for immunoassay.
  • Figure 7A illustrates a sandwich format
  • Figure 7B illustrates a competitive format
  • Figure 8 shows data for hydrogen peroxide determination in the Chamber-Channel unit as illustrated in Figure 4B with a Pt working electrode
  • Figure 9 shows data for glucose determination with glucose oxidase immobilised on a glass bead column in a Chamber ( Figure 5A) and used in a Chamber-Channel unit as illustrated in Figure 4A with a Pt working electrode;
  • Figure 10 shows data for determination of vanadate via enzyme inhibition of alkaline phosphatase.
  • the enzyme was immobilised as in Figure 5A and the Channel used as in Figure 4A with a Au working electrode;
  • Figure 11 obtains the viscosity of samples in the range l-20cP using the Chamber-Channel in Figure 4C with a Au working electrode.
  • Figures 12A to 12D illustrate alternative configurations of chamber.
  • This invention concerns the design of the reservoir or chamber above a flow channel and used together with the channel, the latter containing a flow sensitive sensing area or areas.
  • the linked chamber- channel is a unit in an analytical measurement system.
  • the present invention uses a means for detecting and quantifying an analyte and giving a signal which is a function of sample flow rate.
  • This flow rate dependent detection means may be flow rate dependent due to its comprising a physical or chemical sensor with flow sensitivity (eg an electrochemical or optical sensor etc).
  • the flow rate dependency may arise from an analyte sensitive area (eg enzyme, antibody, analytical reagent) which engages in a reaction with a target component of the sample with reaction turnover dependent on flow.
  • a product or other measurable parameter resulting from that reaction may be determined by a sensor which is not itself flow rate dependent and still provide a detection means which is, as a whole, flow rate dependent.
  • the flow rate dependent means may comprise a means for carrying out a flow rate dependent reaction producing a chemical which is quantified by a non flow-rate dependent sensor for that chemical.
  • the design of the internal profile of the chamber above the channel allows "markers” to be introduced automatically into the signal measured as the sample, which is placed in the chamber, drains into the channel. Markers are preferably discontinuities in the flow-sensitive signal which is measured at the flow rate dependent detection means placed downstream. This results from a disruption or change in the flow from the chamber caused by the shape of the feature designed in the internal profile of the chamber.
  • the markers are not single discontinuities but continuous shape elements, altering the cross-sectional area of the chamber and leading to a resulting change in the flow-sensitive signal.
  • the analytical measurement system disclosed herein comprises a channel with flow rate dependent detection means and a chamber as described below and functions to achieve two key related goals. These goals are, firstly, measurement of the viscosity of a fluid sample and, secondly, measurement of the concentration of an analyte in the fluid sample.
  • the flow rate is linked to the viscosity of the sample, enabling viscosity to be calculated.
  • the flow rate can, in embodiments which require this, be used to calculate the concentration of an analyte measured directly or indirectly by a flow rate dependent detection system. In this case, the flow rate and concentration of the species being detected can all be extracted from the same data signal.
  • a chamber which contains at least one, and preferably two or more, features which change the cross-sectional area of the chamber.
  • the flow rate detection means is flow sensitive, these discontinuities can be detected and so the height of sample in the chamber can be known at that point in time.
  • the chamber delivers a volume or volumes of sample to the flow channel such that the volume flow rate of the delivery can be obtained from the recognisable event marker(s) in the measured signal.
  • the height of the sample drops according to:
  • V f V L dt A (3)
  • A is the cross-sectional area of the reservoir at the top of the solution.
  • the flow rate V f can be obtained directly from the time between two current discontinuities i.e. the time taken for a constant known volume of solution to drain from the reservoir between H 0 and H*.
  • This value can be obtained independently of the magnitude of the measured current values, since it depends only on the time between discontinuities in the current trace and not on the values at those discontinuities.
  • apparatus and a method for measurement of the flow rate of a fluid sample thereby enabling calculation of its viscosity.
  • the flow rate information could be used in the calculation of the concentration of an analyte in the fluid sample.
  • the essential elements are a chamber with at least one discontinuity or other change in cross-sectional area and a channel with a flow-rate dependent detection system.
  • the flow-rate dependent detection system may detect reagent present in the sample or added to the sample by some means.
  • a sample, possibly containing the component to be analysed is placed in a sample chamber 1 having, in this example, two discontinuities in cross-sectional area 2 (giving H 0 ) and 3 (giving H j J.
  • Sample is delivered from the sample to a channel 4 with well-defined flow characteristics and thereafter flows to a sample collection zone 5 which is able to contain the entire volume of the sample when it has drained from a full sample chamber 1 into the channel 4.
  • An optical detection system 6, including source and coupling optics is provided, as is an optical detector 7 such as a photomultiplier tube, photodiode etc.
  • Figure 2 shows a related embodiment in which electrochemical detection is used.
  • the component to be detected is an electrochemically oxidisable or reducible substance.
  • the electrochemical detector comprises a working electrode 8, e.g. Au, Pt, Ag, C etc., a secondary electrode 9, e.g. Pt, C etc and a reference electrode 10 e.g. Ag/AgCl.
  • the electrodes 8-10 are selected depending on a particular species to be measured. In this example, only one discontinuity, 2, is provided, the location if which gives H 0 .
  • the electrochemical detector is used to estimate (eg chronopotentiometrically or coulometrically) the quantity of said oxidisable or reducible substance.
  • the application of the invention includes a fluid flow channel, which contains at least 2 electrodes, each electrode being in electrical contact with the channel.
  • the channel is integrated with a sample supply chamber, which has an internal profile having at least one change in cross sectional area ( Figures 2 and 3). The sample is placed in the chamber and it flows from the chamber into the channel. As the sample passes the electrodes, an electrochemical signal is generated which is sensitive to flow rate.
  • the viscosity of the sample can be calculated.
  • the flow rate dependent measurement of a chemical species can now be used to calculate the concentration of a chemical species as the flow rate is now known.
  • the chemical species may be an analyte in the original sample or which has a concentration linked in some way with the concentration of analyte in the original sample.
  • the chemical species which is measured is produced within the analytical sample at a reaction zone.
  • One example comprises placing the sample, possibly containing component to be analysed, in the chamber from where it is delivered to a reaction zone sensing area and thence to an electrode sensing area in the channel, and allowing it to produce directly or indirectly a corresponding quantity of an electrochemically oxidisable or reducible substance into said channel at the reaction zone and then electrochemically estimating the said oxidisable or reducible substance in said channel at the electrode as a measurand of the quantity of said component to be measured.
  • the said reaction zone comprises a thin layer of reaction component(s), overlaying the channel wall, upstream of the electrode sensing area; the said electrode sensing area comprising at least two electrodes as described also above.
  • the said reaction zone comprises reaction component loaded onto a solid support, and filling across the channel at or near the base of the reservoir.
  • the reaction components include chemical and biochemical reagents eg enzymes, cells, metals, redox reagents.
  • the measurement of a component of a liquid system comprises placing the sample, possibly containing component to be analysed, in the chamber from where it is delivered to a reaction zone sensing area and thence to an optical sensing area in the channel (figure 1), and allowing it to produce directly or indirectly a corresponding quantity of a substance which exhibits light-absorbing or luminescence and fluorescence properties into said channel at the reaction zone and estimating the said substance in said channel at a detector (eg photodiode) as a measurand of the quantity of said component to be measured.
  • the material from which the channel is formed is transparent and optically coupled to the photodetector and light source (where required) with suitable components (eg prisms, lens, mirrors).
  • the invention also provides for the use of multiple chambers used consecutively or in tandem with one channel or several channels.
  • amperometric electrodes may be placed in the flow channel and a method is provided whereby the viscosity of a sample may be measured.
  • the viscosity of the sample will influence both flow rate and diffusion of species to the electrode.
  • the diffusion coefficient, D which expresses this is a factor of the Levich equation (equation (1)) and is related to the viscosity, ⁇ , using the Stokes-Einstein equation:
  • k will be constant for a particular flow cell, electroactive species and concentration.
  • the magnitude of the signal measured by the flow rate dependent detection means is not of interest; only the time variation of the signal is required for flow and/or viscosity measurement.
  • the invention also encompasses a computer program to calculate the flow rate, viscosity or analyte concentration of a sample.
  • the computer program takes a time course of readings from the flow rate dependent detection means and uses the above techniques to establish when the fluid sample passed one or more features in the cross-section of the chamber. Using the known dimensions of the chamber, the flow rate or viscosity of the fluid sample can then be calculated. In embodiments where analyte concentration is to be measured, the computer program then use the known flow rate or viscosity to calculate analyte concentration using readings from the flow rate dependent detection means and using the known flow rate / viscosity.
  • the invention also extends to computers adapted to perform the computer program, computer programs stored on or in a carrier, adapted for putting the invention into practice.
  • the program may be in the form of source code, object code, a code intermediate source and object code such as in a partially compiled form, or in any other form suitable for implementation of the processes according to the invention.
  • the carrier may comprise a storage medium, such as a ROM, CD-ROM, semiconductor rom, programmable or field programmable semiconductor, PIC, EEPROM, magnetic recording medium or may be a transmissible carrier such as an electrical or optical signal conveyed via electrical or optical or radio means.
  • the Channel and Chamber In the example given in figure 1 and figure 2, the channel unit has dimensions of 15mm x 2mm. The depth of the channel is ⁇ 0.3mm. These are typical but not exclusive dimensions to achieve laminar flow. Laminar flow is preferred but by no means essential.
  • the channel passes into a collection chamber, which is continuous with the channel, having such volume as able to contain said sample in its entirety ( Figure 1 B). According to this example, the channel-chamber unit is limited to one sample, after which it must be emptied or disposed.
  • the outflow is guided through an outflow port 1 1 down a rod 12 (diameter 0.8 mm held in a block in this example) into a lower waste collector 13. According to this example, there is no limit on the number of samples which can pass through the chamber and channel and be collected in the waste.
  • An open-flow channel may also be substituted.
  • the channel Above the channel is a chamber having as a minimum, one discontinuity in the internal diameter at height H 0 above the channel base, as shown in figure 2.
  • Sample volume measurement is not required, but sample is filled to above the discontinuity.
  • the 'discontinuity' in the internal profile of the chamber must cause a change in flow rate as the sample drains from the chamber.
  • Designs causing a change in flow include orifices, nozzles, cones and baffles. In a preferred but not exclusive embodiment (figure 2) this is achieved by a step change in the cross sectional area.
  • the channel unit has the same dimensions as in figure 2 but the chamber above the channel had two discontinuities at heights H 0 and Hi as shown in figure 1.
  • the volume contained in the chamber between these two heights is a constant given by ⁇ (A*/2) 2 (H 0 - H,), where A, is the cross- sectional area in the chamber between the discontinuities.
  • the channel and chamber may be oriented in any direction. It will be clear to one skilled in the art that the chamber need not be vertically oriented but may be positioned in any orientation provided that there is some vertical height difference between the sample and the outflow to drive fluid flow.
  • the chambers) with the internal profile leading to flow measurement is produced as a separate unit to the channel (figure 5), the latter containing a detection system.
  • the chamber(s) may be provided integral to the channel, or adapted to be joined to the channel. The invention allows for several chambers to be used together.
  • the channel is shown as horizontal in the accompanying diagrams, it will be recognised by one skilled in the art that it could be positioned in any orientation including being vertical, parallel with the axis of the chamber.
  • Figure 12A illustrates a chamber and channel where the chamber and channel are vertical and coaxial, with an integrated waste collector and counter electrode functioning as a flow restricting means.
  • Figure 12B illustrates a channel with three step changes in cross-sectional area. Any number of step changes can be provided, indeed, the additional of further step changes allows additional data points to be taken into account to provide a more accurate final measurement.
  • Figure 12C illustrates a chamber having four step changes. Here, the cross section narrows, increases and narrows again twice as fluid drains from the chamber, providing additional data points.
  • Figure 12D illustrates a vertical integrated chamber and channel where individual regions have a curved profile.
  • Figure 5A shows a possible but not exclusive position in a two-step chamber unit for reagents in an analyte sensitive area (eg enzyme, antibody, analytical reagent) which engages in a reaction with a target component of the sample.
  • analyte sensitive area eg enzyme, antibody, analytical reagent
  • This chamber unit is 'plugged' into a channel unit during the analytical measurement procedure and liquid added to the chamber to a level above the top discontinuity in the profile, drains into the channel.
  • the chamber unit has a reagent zone sensing area, typically but not exclusively involving immobilised reagent on beads.
  • Figure 5B shows two reagent containing chambers and reagent zone sensing areas 17a, 17b used in serial; the tandem may be plugged into the channel at the start of the procedure or part of the analytical procedure completed before linking to the channel either together or separately.
  • Figure 5C introduces an additional sampling column, plunger or syringe 18, said unit which may also contain reagent to capture or react with a component of the sample to be measured.
  • the walls of the sampling column, plunger or syringe 18 may be modified with capture ligand 19 for the target analyte.
  • Reagents may also be placed in a sensing area in the channel, upstream of the detector (eg Figure 6) in which there is a sensing area 20 with immobilised reagent at the entry to the Channel and a second sensing area 21 on the same or opposite sides of the Channel. Their position and geometry will affect the resulting flux patterns.
  • FIG. 4 shows possible but not exclusive position(s) for the reducer.
  • a preferred but not exclusive form for the reducer is a porous plug 14 or 15 at one end of the channel 16 ( Figures 4A and 4B); in an alternative design a rod or tube annulus is introduced into the chamber ( Figure 4C). In the latter case the tube annulus may also serve to deliver reagents or wash to the channel.
  • a flow reducer may also be implemented by simply having a narrow portion of the channel or using a capillary tube and, in general, the narrowest part of the fluid flow route will limit flow rate.
  • a detector is placed downstream of the chamber, usually in the channel. In the absence of other 'sensing areas' the detector should give a signal which shows flow sensitivity. Electrochemical measurements are an example of such a measurement. In Figure 2, three electrodes are incorporated in the channel in contact with the fluid flowing through the channel. Electrochemical measurements can be made with this channel. In the presence of 'sensing areas' having a reaction with a component of the sample, with a turnover rate dependent on flow of a compound detected downstream, the detector placed downstream of this sensing area may also give a signal dependent on flow. Simple optical measurements are an example of such a measurement. In figure 1 , the material from which the channel is formed is transparent and optically coupled to the photodetector and light-source.
  • the detector could also function as the flow rate reducer.
  • an appropriate metal rod could function both as an electrode for an electrochemical sensor and as a flow rate reducer.
  • an optical fibre or other optical component could fulfil this function. Where the reaction zone has a solid support, this could also be configured to function as a flow rate reducer.
  • the channel can be manufactured as a single or several component system. Typically, but not exclusively it may comprise an open channel or channel unit and coverplate. Overall reduction in flow is achieved with a porous plug. If the channel is to be used for multiple samples, the outflow is guided down a rod (diameter 0.8 mm held in a block) into a lower waste collector (figure 3), else it is contained in a collection chamber which is continuous with the channel (figure 1).
  • Flow in the channel is characterised using a chamber having one or two discontinuities in the internal diameter as shown in figures 2 and 3. Analysis is performed by filling the chamber with sample. Exact sample volume measurement is not required. Nominal solution height in the chamber is measured from the base of the porous plug.
  • the channel contains 3 electrodes, each being in contact with the fluid passing through the channel. All potentials are measured relative to a standard Ag/AgCl reference electrode place on one wall of the channel. A working electrode polarised at a potential such that all electroactive species to be measured arriving at it are oxidised or reduced is placed on the opposite wall, together with a counter electrode.
  • the desired analyte is hydrogen peroxide: hydrogen peroxide is monitored by its oxidation current on a Pt electrode at +600mV vs Ag/AgCl.
  • a chamber having one discontinuity in its internal profile at height H 0 is used (figure 2).
  • the chamber is filled with sample and begins to drain into the channel, where a current due to the hydrogen peroxide in the sample is measured.
  • This sudden change in current marks I 0 at H 0 and hence V f can be estimated to be able to obtain the concentration information from equation (1), the current must be taken at the point where the sample in the reservoir passes H 0 .
  • a chamber having two discontinuities in its internal profile at heights H 0 and Hi is adopted.
  • the chamber is filled with sample and the sample drains into the channel where the electrochemical oxidation of hydrogen peroxide is measured.
  • the cell reservoir volume between the two discontinuities in the chamber
  • V f the draining of this volume of solution is marked by two features in the current trace produced when the sample meniscus passes the discontinuities; thus V f can be calculated from the time between the current markers.
  • the current measured from a sample added to this chamber-channel unit can calibrated for concentration: this leads to the curve shown in figure 8.
  • a reagent loaded column is incorporated placed between the chamber reservoir and channel, said column being typically but not exclusively porous glass.
  • the reagent is an enzyme, glucose oxidase
  • the analyte is glucose
  • analyte is hydrogen peroxide.
  • the concentration of glucose is related to the concentration of hydrogen peroxide.
  • the measurement of hydrogen peroxide is achieved by further chemical reaction with a chromogenic or fluorogenic reagents such as luminol, peroxidase/ aminophenazone to produce a species which may be detected optically, in which case the channel illustrated by example in figure 1 is used. Alternatively, it may be determined electrochemically as described in the example above.
  • the chamber-channel unit shown in figure 4A may be used.
  • a porous plug is used to slow the overall flow, placed between the chamber and channel.
  • This is the same chamber-channel unit employed as for hydrogen peroxide determination, together with the incorporation of an enzyme column.
  • porous plugs were cut from 2.7 mm thick coarse grade (90 - 150 ⁇ m) hydrophilic porous polyethylene, to fit the bottom of the 2mm diameter tube connecting the chamber to the flow channel.
  • the enzyme was loaded onto controlled-pore glass beads of size 125-177 ⁇ m which had been amino- silylated by immersion for 2 minutes in a 5 minute-old mixture of 3-aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml).
  • glucose oxidase (GOD) (type VII from aspergillus niger) could be loaded onto the aminated surface using cobalt ions": cobalt (II) chloride hexahydrate (40 mg) and glucose oxidase (20 mg) were dissolved in water (6ml) and amino-silylated glass beads (200 mg) were immediately added. The whole was stirred for 10 minutes then filtered to separate the beads, which were dried in air at room temperature. Exposure to air allowed the cobalt to oxidise to the kinetically inert Cobalt (III).
  • cobalt ions cobalt ions
  • poly(glycidyl)methacrylate was precipitated from benzene (1 ml of 10 % solution) onto the aminosilylated glass beads (100 mg) which were suspended in stirred methanol (10 ml). GOD could then be loaded onto the polymer surface" 1 : PGMA-coated beads (15 mg) were shaken with GOD (1.5 mg) in borate buffer (1.5 ml of 0.05M sodium borate, pH 8.5) for 24 hours, isolated by filtration, rinsed in water, phosphate buffer, and water again, and dried in air.
  • borate buffer 1.5 ml of 0.05M sodium borate, pH 8.5
  • Sample containing the analyte of interest, flows from the chamber into the channel, over the enzyme column where the analyte reacts with the enzyme.
  • the analyte glucose and of glucose oxidase enzyme reagent the hydrogen peroxide produced in the enzyme reaction is swept down the channel by the solution flow and is detected at the downstream detector.
  • the cell reservoir volume between the two discontinuities in the chamber
  • V the draining of this volume of solution is marked by two features in the current trace produced irrespective of analyte concentration, when the sample meniscus passes the discontinuities; thus V can be calculated from the time between the current markers.
  • the channel shown in figure 3 was used. 3 electrodes where included in the channel during manufacture: A Pt working electrode, a Pt counter electrode and a Ag/AgCl reference electrode. The working electrode was poised at 0. IV vs Ag/AgCl using a potentiostat to measure the electrochemically oxidisable product of the enzyme reaction. Chronoamperometric data from the flow cell were collected for each sample. The channel-chamber unit was used in high through-put 'batch' mode by refilling the reservoir ( ⁇ 200 ⁇ l) each time it drained below the bottom chamber discontinuity.
  • a porous plug is used, to slow the overall flow, placed between the chamber and channel.
  • An enzyme loaded column in used as described for the previous example for the determination of glucose.
  • the porous plugs were cut from 2.7 mm thick coarse grade (90 - 150 ⁇ m) hydrophilic porous polyethylene, to fit the bottom of the 2mm diameter tube connecting the chamber to the flow channel.
  • the enzyme was loaded onto controlled-pore glass beads of size 125-177 ⁇ m which had been amino-silylated by immersion for 2 minutes in a 5 minute-old mixture of 3- aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml).
  • poly(glycidyl)methacrylate (PGMA) was precipitated from benzene (1 ml of 10 % solution) onto the aminosilylated glass beads (100 mg) by slow addition of the benzene solution to a rapidly stirred suspension of the glass beads in methanol (10 ml).
  • Alkaline phosphatase could then be loaded onto the polymer surface"": PGMA-coated beads (1 g) were shaken with a solution of ALP (10,000 units, as supplied) in diethanolamine buffer (5 ml of 1 M diethanolamine, 0.5 mM magnesium chloride, 0.1 M potassium chloride, pH 9.8) for 48 hours at 4 °C, isolated by filtration, rinsed in buffer and then distilled water (x3) and dried in air, all at 4 °C. 4mg of enzyme loaded glass beads were placed above the porous plug in the region at the base of the chamber, below the lower discontinuity. The amount of enzyme on a column is kept low (compared with glucose determination method).
  • the enzyme column therefore converts only a small fraction of the substrate passing through it, and so shows the expected simple inverse relationship between concentration and flow rate.
  • the enzyme activity in the plug can thus be determined.
  • Typical but not exclusive potential substrates for this enzyme are the phosphates of naphthol, vanillin, 4-methylaminophenol sulfate, 4-aminophenol, hydroquinone, which produce an electrochemically oxidisable or reducible product of the enzyme reaction.
  • the presence of an inhibitant in the sample is measured by mixing the said sample with the phosphate substrate for the enzyme and adding ⁇ 200 ⁇ L into the chamber.
  • the following estimation of the inhibition of conversion of the phosphate by the enzyme to the electrochemically oxidisable product is obtained from the current measured at the channel electrodes.
  • Two types of information can be obtained by comparing the activity of the enzyme in the absence of inhibitant with the activity of a sample possibly containing inhibitant: the rate of inhibition of the enzyme and the equilibrium turnover of the enzyme.
  • V f Thas the units of mole per second when c ⁇ is expressed in moles/dm 3 and. V m cm 3 /s.
  • the flow rate is determined from the time between the two constriction events. Typical data for the inhibition of ALP by vanadate is given in figure 10.
  • a channel-chamber unit such as figure 1, figure 3 or figure 4C: said chamber having two steps (sequential discontinuities) defining a volume of sample and the draining of this volume of solution is marked by two features in the current trace produced when the sample meniscus passes the discontinuities.
  • V f can be calculated from the time interval between these current markers and is related to sample viscosity according to equation (12).
  • Plugs were made from 2.7 mm thick coarse grade (90 - 150 ⁇ m) hydrophilic porous polyethylene, cut to fit the bottom of the 2 mm diameter tube connecting the chamber to the flow channel.
  • the annulus was constructed by placing a 1.6 mm external diameter rod or tube along the axis of the reservoir and extending it along the length of the 2 mm tube connecting the reservoir to the flow cell. It was maintained in a central position by supports. The overall reduction in flow provided by this constriction is expressed by the formula:
  • the necessary electrode current signal may be obtained by adding an agent to the sample which may be electrochemically oxidised or reduced. However, in most cases this is unnecessary with aqueous samples containing dissolved oxygen, since the electrochemical reduction of oxygen may be used to provide the marker signal. Concentration is not obtained here from the data (although it could be if required), but the current only used as a marker of flow.
  • the channel required is the same as described above for enzyme inhibition data, except a Au working electrode is employed in this example. Chronoamperometric data from the flow cell is collected at -0.6 V vs Ag/AgCl where oxygen is reduced. The channel-chamber unit was used in high through-put 'batch' mode by refilling the reservoir ( ⁇ 200 ⁇ l) with a new sample each time it drained below the bottom chamber discontinuity.
  • the sample (solutions of glycerol having viscosity in the range 1 - 20cP) is added to the chamber ( ⁇ 200 ⁇ L) and the current monitored due to the electrochemical reduction of dissolved oxygen. Discontinuities are observed in the current signal (current markers) as the sample meniscus passes the discontinuities in the cross-sectional area of the chamber. Obtaining flow rate V f , from the time interval between current markers gives a linear relationship of a wide range of viscosities (see figure 1 1).
  • Chamber la has a reagent column 22 having typically, but not exclusively, immobilised reagent on beads; the reagent being selected to have affinity for the target antigen analyte.
  • the column 22 was prepared with IgG antibody against an epitope of the target component of the sample. The antibody was loaded onto control led-pore glass beads of size 125-177 ⁇ m which had been amino-silylated by immersion for 2 minutes in a 5 minute-old mixture of 3-aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml). The methods used are the same as for loading with enzyme as described above.
  • Chamber lb has a reagent column prepared with an antibody to the labelled antibody 24. Chamber lb may be 'plugged' into the channel at the beginning of the procedure or else before the final measurement step. Chamber la is 'plugged' into Chamber lb. Sample of known volume which might contain target antigen is added to Chamber la and drained to waste. Antigen present in the sample is captured in Chamber 1 a according to the binding equilibrium.
  • Labelled sandwich antibody of known concentration and volume in added to Chamber la and drained through Chamber lb to waste where 25 is reagent zone sensing area 22 after capture of any sample antigen 23 and 26 is a reagent- zone sensing area typically but not exclusively involving immobilised reagent on beads; this reagent has an affinity for the labelled antibody.
  • 27 is zone 26 after capture of any labelled antibody
  • the labelled antigen is captured in Chamber la and Chamber lb relative to the amount of sample antigen that has been bound in the first step in Chamber la. Chamber la is separated from Chamber 1 b, the latter remaining plugged into the channel, and enzyme substrate added ⁇ 150 ⁇ L. The enzyme turnover of substrate is estimated at the electrode in the channel as for the toxicity test above.
  • Chamber la can be plugged into a channel and an estimate of bound sandwich enzyme made by the same method, thus giving a ratio of enzyme label bound in each Chamber.
  • Figure 7B illustrates a similar procedure for competitive immunoassay in which labelled antigen 28 is added to the sample which may already contain a concentration of antigen 23.
  • the reagent zone 22 captures a mixture of sample antigen 23 and labelled antigen 28, giving zone 29. Residual labelled antigen is captured in reagent zone 30, giving zone 31.
  • Enzyme substrate is added as in the previous example, leading to a measurement of bound labelled antigen from which the concentration of antigen in the original sample can be calculated.
  • Ada 281 (1993) 645-653.

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Abstract

L'invention concerne un capteur destiné à quantifier le débit ou la viscosité d'un échantillon fluidique, ou la concentration d'un analyte dans cet échantillon fluidique. Ce capteur comprend une chambre destinée à loger un échantillon fluidique et à le guider à travers un canal contenant un dispositif de détection. Cette chambre comprend au moins une et de préférence deux variations de diamètre entraînant des changements détectables dans l'écoulement de l'échantillon dans la chambre et dans les mesures du dispositif de détection. Les dimensions de la chambre étant connues, on peut calculer le débit et la viscosité de l'échantillon fluidique. Ces résultats peuvent alors être utilisés pour calculer la concentration d'un analyte, directement ou indirectement, en dépit de toute dépendance de débit du dispositif de détection.
PCT/GB2001/001650 2000-04-12 2001-04-12 Appareil et procede destines a l'analyse d'un analyte fluidique WO2001076743A2 (fr)

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Cited By (2)

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CN103439260A (zh) * 2013-09-17 2013-12-11 国核电站运行服务技术有限公司 分光光度计固体样品支架
EP2212704A4 (fr) * 2007-11-09 2015-06-03 Canon Kk Mécanisme d'entraînement d'alimentation en liquide utilisant une pompe osmotique et micropuce dotée du mécanisme d'entraînement d'alimentation en liquide

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CN107389911A (zh) * 2017-07-12 2017-11-24 柳州康云互联科技有限公司 用于移动端的快速分离检测传感器

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DE3138514A1 (de) * 1981-09-28 1983-04-14 Klaus Dipl.-Ing. 5100 Aachen Mussler Verfahren und vorrichtung zur bestimmung des fliessverhaltens biologischer fluessigkeiten
US4793174A (en) * 1987-10-05 1988-12-27 E. I. Du Pont De Nemours And Company Differential pressure capillary viscometer
US5486335A (en) * 1992-05-01 1996-01-23 Trustees Of The University Of Pennsylvania Analysis based on flow restriction
WO1999009388A2 (fr) * 1997-08-18 1999-02-25 Metasensors, Inc. Procede et appareil pour l'analyse de gaz en temps reel
WO2001024297A1 (fr) * 1999-09-24 2001-04-05 Siemens Aktiengesellschaft Determination de la concentration d'alcool dans l'electrolyte de piles a combustible

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Publication number Priority date Publication date Assignee Title
DE3138514A1 (de) * 1981-09-28 1983-04-14 Klaus Dipl.-Ing. 5100 Aachen Mussler Verfahren und vorrichtung zur bestimmung des fliessverhaltens biologischer fluessigkeiten
US4793174A (en) * 1987-10-05 1988-12-27 E. I. Du Pont De Nemours And Company Differential pressure capillary viscometer
US5486335A (en) * 1992-05-01 1996-01-23 Trustees Of The University Of Pennsylvania Analysis based on flow restriction
WO1999009388A2 (fr) * 1997-08-18 1999-02-25 Metasensors, Inc. Procede et appareil pour l'analyse de gaz en temps reel
WO2001024297A1 (fr) * 1999-09-24 2001-04-05 Siemens Aktiengesellschaft Determination de la concentration d'alcool dans l'electrolyte de piles a combustible

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2212704A4 (fr) * 2007-11-09 2015-06-03 Canon Kk Mécanisme d'entraînement d'alimentation en liquide utilisant une pompe osmotique et micropuce dotée du mécanisme d'entraînement d'alimentation en liquide
US9339814B2 (en) 2007-11-09 2016-05-17 Canon Kabushiki Kaisha Liquid supply drive mechanism using osmotic pump and microchip having the liquid supply drive mechanism
CN103439260A (zh) * 2013-09-17 2013-12-11 国核电站运行服务技术有限公司 分光光度计固体样品支架

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